WO2020155141A1 - 治疗神经退行性疾病或rna结合蛋白发生异常导致的疾病的药物组合物及其应用 - Google Patents

治疗神经退行性疾病或rna结合蛋白发生异常导致的疾病的药物组合物及其应用 Download PDF

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WO2020155141A1
WO2020155141A1 PCT/CN2019/074579 CN2019074579W WO2020155141A1 WO 2020155141 A1 WO2020155141 A1 WO 2020155141A1 CN 2019074579 W CN2019074579 W CN 2019074579W WO 2020155141 A1 WO2020155141 A1 WO 2020155141A1
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hnrnp
tdp
rna binding
pharmaceutical composition
protein
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PCT/CN2019/074579
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French (fr)
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方燕姗
段永嘉
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中国科学院上海有机化学研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/502—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the invention relates to a pharmaceutical composition for treating neurodegenerative diseases or diseases caused by abnormalities in RNA binding proteins and applications thereof.
  • ALS Amyotrophic lateral sclerosis
  • ALS is a neurological disease caused by the degeneration and degeneration of motor neurons, with an average of 1-2 cases per 100,000 people (Bento-Abreu et al., 2010). ALS disease is mainly manifested by the massive loss of descending motor neurons (including brain stem and ventral horn) and ascending motor neurons (including cortex, etc.). The main symptoms include: muscle atrophy, muscle spasm, dysarthria, dysphagia, and In the later stage, the patient loses basic exercise ability and will eventually die due to respiratory failure (Mitchell and Borasio, 2007; Nguyen et al., 2018; van Es et al., 2017).
  • ALS disease is still incurable.
  • the only clinical drugs approved by the US Food and Drug Administration (FDA) are Riluzole and Edaravone.
  • Riluzole slows down the progression of ALS by inhibiting glutamatergic activity, but the drug has poor specificity and can only prolong the life of the patient for 3-6 months, and is accompanied by nausea, dizziness and even pneumonia.
  • Side effects (Lacomblez et al., 1996; Tripathi and Al-Chalabi, 2008).
  • Edaravone is a free radical scavenger. Phase III clinical results show that Edaravone can relieve the physical damage of ALS patients to a certain extent.
  • RNA binding proteins function by binding to RNA to form ribonucleoprotein (RNP) complexes.
  • RNP ribonucleoprotein
  • RNP complexes form stress granules to maintain cell homeostasis (Buchan et al., 2009).
  • the genes encoding these proteins are mutated, they can affect the formation and function of RNP particles and promote their abnormal aggregation, which is an important pathological sign of a variety of neurodegenerative diseases, including ALS.
  • RNA binding proteins There are hundreds of RNA binding proteins in cells.
  • RNA binding proteins related to ALS disease mainly include heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), Transactivation response element DNA-binding protein 43 (TDP-43), Fused in sarcoma ( FUS), TATA-binding protein-associated factor 15 (TAF15), Ewing sarcoma breakpoint region 1 (EWSR1), T-cell intracytoplasmic antigen 1 (TIA-1), etc. (Harrison and Shorter, 2017).
  • hnRNP A1 and TDP-43 are mainly located in the nucleus under normal physiological conditions and participate in the regulation of various cellular physiological functions.
  • hnRNP A1 and TDP-43 proteins include: (1) Abnormal post-translational modifications of hnRNP A1 and TDP-43 proteins, such as hyperphosphorylation, ubiquitination, etc. and abnormal intracellular aggregation (Neumann et al. , 2006).
  • hnRNP A1 and TDP-43 proteins were found to be abnormally localized in the cytoplasm and form pathological inclusion bodies (Kim et al., 2013; Neumann et al., 2006).
  • the formation and regulation of stress granules involved in hnRNP A1 and TDP-43 are abnormal: hnRNP A1 and TDP-43 proteins are both important components of cell stress granules; hnRNP A1 and TDP-43 gene mutations can Affect their ability to locate and bind to stress particles; accordingly, regulating the formation of stress particles can alleviate the neurotoxicity caused by these RNA binding proteins (Elden et al., 2010; Liu-Yesucevitz et al., 2010) ;Li et al., 2013; Kim et al., 2013; Kim et al., 2014; Naruse et al., 2018).
  • the molecular mechanisms of hnRNP A1 and TDP are abnormally localized in the
  • RNA binding proteins such as hnRNP A1 can interact with RNA to form RNP particles, especially cell stress particles.
  • RNA-binding proteins can form droplets similar to stress particles in cells through Liquid-Liquid Phase Separation (LLPS) in vitro experiments (Lin et al., 2015). These droplets are highly dynamic and reversible, and can fuse or separate with other droplets around.
  • LLPS Liquid-Liquid Phase Separation
  • the droplets formed by LLPS continue to solidify and gradually form amyloid aggregates (Hyman et al., 2014; Lin et al., 2015; Molliex et al., 2015; Wang et al., 2014) .
  • This process is similar to the process that stress particles in the body gradually lose fluidity and reversibility in the process of ALS disease, and eventually develop into irreversible pathological protein inclusion bodies, so it can be used to characterize the tendency of RNA binding proteins to aggregate.
  • PARylation The reversible reaction of protein PARylation modification is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of polymerases and the hydrolase poly(ADP-ribose) glycohydrolase (PARG).
  • PARP poly(ADP-ribose) polymerase
  • PARG hydrolase poly(ADP-ribose) glycohydrolase
  • the cell functions involved in the regulation of PARylation modification mainly include: (1) Participate in regulating chromatin structure and gene transcription; (2) Assist in the assembly of DNA damage repair complex and promote its function; (3) Activate the proteasome to remove damaged histones ; (4) Promote the transport of proteins into the Cajal body; (5) Influencing the nuclear transport of proteins; (6) Promote the mitochondria to release apoptosis inducing factors (AIF) and their transport to the nucleus; (7) Participate in the regulation of the assembly and normal function of stress particles (Luo and Kraus, 2012).
  • PARP inhibitors The clinical application and drug development of PARP inhibitors is mainly focused on tumor treatment.
  • the PARP1 inhibitor Olaparib was approved by the FDA for ovarian cancer, breast cancer, and prostate cancer related to BRCA1 and BRCA2 gene mutations. Cancer treatment (Fong et al., 2009; Tutt et al., 2010).
  • a small number of laboratory and pre-clinical studies suggest that reducing the level of PARylation also has a certain neuroprotective effect on Huntington's disease, cerebral ischemia and axonal damage (Brochier et al., 2015; Cardinale et al., 2015; Egi et al., 2011; Teng et al., 2016).
  • PARP inhibitors such as Olaparib can be used in the treatment of diseases such as ALS.
  • Tankyrase (PARP5) is the main enzyme that catalyzes the modification of TDP-43PARylation; Tankyrase is mainly located in the cell cytoplasm.
  • the Tankyrase inhibitors XAV939 and G007-LK used in the study of McGurk et al. were not FDA-approved clinical drugs and were only inhibitors for laboratory research; and XAV939 and G007-LK were not used to inhibit TDP in their study. -43 initiated the effectiveness and safety experiments on neurotoxicity.
  • the technical problem to be solved by the present invention is to overcome the lack of effective drugs for treating neurodegenerative diseases or diseases caused by abnormalities of RNA binding proteins in the prior art, and provide a treatment for neurodegenerative diseases or abnormalities of RNA binding proteins.
  • the pharmaceutical composition and application thereof especially in the treatment of amyotrophic lateral sclerosis (ALS) disease.
  • ALS amyotrophic lateral sclerosis
  • RNA binding proteins such as hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) and TDP-43 (Transactivation response element DNA-binding protein 43) proteins
  • the dynamic nature of the main purpose is to inhibit the assembly of stress particles and/or promote the disassembly of stress particles; affect the interaction between RNA binding proteins such as hnRNP A1 and other poly-ADP ribosylated proteins; affect RNA binding The interaction between proteins such as hnRNP A1 and other PAR binding proteins; affect the subcellular localization and stress response of RNA binding proteins such as hnRNP A1; affect the liquid-liquid phase separation and aggregation tendency of RNA binding proteins such as hnRNP A1; affect RNA Co-phase separation of binding proteins such as hnRNP A1 and TDP-43 protein; affecting the interaction of RNA binding proteins such as hnRNP A1
  • PARP inhibitors are mostly used in tumor treatment, and there is no research or report showing that drugs that reduce the level of poly-ADP ribosylation modification in cells, such as Olaparib, can be used in the treatment of neurodegenerative diseases such as ALS.
  • the present invention is the first to conduct ALS disease treatment experiments on a small molecule inhibitor of PARP approved by the FDA for tumor treatment, Olaparib, and find that the inhibitor has obvious neurotoxicity induced by hnRNP A1 and TDP-43 Inhibition, so the inhibitor can be used to develop clinical drugs for the treatment of ALS and other related diseases.
  • the present invention provides a pharmaceutical composition for the treatment of neurodegenerative diseases.
  • the pharmaceutical composition includes a drug that reduces the level of intracellular poly-ADP ribosylation modification, and preferably reduces intracellular RNA-binding protein. Poly ADP ribosylation modified level of drugs.
  • the RNA binding protein is hnRNP A1 and/or TDP-43.
  • the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • the present invention provides a pharmaceutical composition for the treatment of diseases caused by abnormalities in RNA binding proteins.
  • the pharmaceutical composition includes a drug that reduces the level of poly ADP ribosylation modification in cells, preferably reducing intracellular
  • the RNA binding protein has a modified level of poly ADP ribosylation; the RNA binding protein is preferably hnRNP A1 and/or TDP-43.
  • the disease caused by the abnormality of the RNA binding protein is amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • RNA binding proteins Preferably, the treatment of diseases caused by abnormalities in RNA binding proteins includes one or more of the following:
  • RNA binding proteins Preferably, the treatment of diseases caused by abnormalities in RNA binding proteins includes one or more of the following:
  • RNA binding protein such as hnRNP A1
  • covalent poly ADP ribosylation modification of RNA binding protein such as hnRNP A1 affects the interaction and/or subcellular localization of the RNA binding protein such as hnRNP A1 with other proteins such as TDP-43 protein;
  • RNA-binding proteins such as hnRNP A1 affect the interaction of the RNA-binding proteins such as hnRNP A1 with other proteins such as TDP-43 protein through non-covalent binding to PAR.
  • RNA-binding protein affects its solubility in cells by non-covalently binding PAR, thereby causing diseases caused by the destruction of cell homeostasis.
  • the drug for reducing the level of poly ADP ribosylation modification in a cell in the present invention is a drug that can increase the expression level of PARG hydrolase and/or reduce the expression level of PARP polymerase;
  • the drug capable of increasing the expression level of PARG hydrolase is a PARG hydrolase agonist
  • the drug capable of reducing the expression level of PARP polymerase is a PARP polymerase inhibitor; the PARP polymerase inhibitor is preferably Olaparib.
  • the present invention provides a method for treating the above-mentioned neurodegenerative diseases or diseases caused by the abnormality of the above-mentioned RNA binding protein, the method comprising using the above-mentioned pharmaceutical composition for treatment.
  • the pharmaceutical composition is treated by one or more of the following mechanisms:
  • the pharmaceutical composition can ultimately inhibit neurodegeneration caused by hnRNP A1 and/or TDP-43.
  • the neurodegeneration is preferably mainly manifested as: 1) toxicity inhibition in motor neuron-like NSC-34 cells, and/or, 2) neuronal degeneration, reduced exercise capacity and lifespan in the Drosophila model of ALS disease The alleviating effect of shortening and other phenotypes.
  • the present invention provides the application of the above-mentioned pharmaceutical composition in the treatment of the above-mentioned neurodegenerative diseases or diseases caused by the abnormality of the above-mentioned RNA binding protein.
  • the present invention provides the application of the above-mentioned pharmaceutical composition in the preparation of drugs for the treatment of the above-mentioned neurodegenerative diseases or diseases caused by the abnormality of the above-mentioned RNA binding protein.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the positive and progressive effect of the present invention is that the pharmaceutical composition of the present invention such as Olaparib can significantly enhance the dynamics of stress particles containing RNA binding proteins such as hnRNP A1 and TDP-43 proteins, mainly by inhibiting the assembly and/or Promote the disassembly of stress particles; affect the interaction between RNA binding proteins such as hnRNP A1 and other poly-ADP ribosylated proteins; affect the interaction between RNA binding proteins such as hnRNP A1 and other PAR binding proteins; Influencing the subcellular localization and stress response of RNA binding proteins such as hnRNP A1; affecting the liquid-liquid phase separation and aggregation tendency of RNA binding proteins such as hnRNP A1; affecting the co-phase separation of RNA binding proteins such as hnRNP A1 and TDP-43 proteins; Affect the interaction of RNA binding proteins such as hnRNP A1 and TDP-43 protein in cells.
  • RNA binding proteins such as hnRNP
  • Figure 1 shows the results of the dynamic process of the PARylation level affecting the assembly-disassembly of stress particles containing hnRNP A1 protein in Example 1.
  • Figure 2 shows the results of the dynamic process of the PARylation level affecting the assembly-disassembly of TDP-43 protein-containing stress particles in Example 2.
  • Figure 3 shows the results of the hnRNP A1 protein in Example 3 that can not only undergo PARylation, but also bind to PAR.
  • Figure 4 shows the results of PARylation or PAR binding in Example 4 that can affect the subcellular localization and stress response of hnRNP A1.
  • Fig. 5 shows the result of PARylation capable of regulating the interaction between hnRNP A1 and TDP-43 in Example 5.
  • Figure 6 shows the result of PAR in Example 6 that can promote the liquid-liquid phase separation of hnRNP A1 in vitro.
  • Fig. 7 shows the result of hnRNP A1 and TDP-43 in Example 7, and PAR can promote this process.
  • Figure 8 shows the result of cytotoxicity caused by overexpression of hnRNP A1 or TDP-43 in motor neuron-like NSC-34 cells in Example 8.
  • Figure 9 shows the results of the PARylation level in Example 9 that can regulate the cytotoxicity caused by hnRNP A1 and TDP-43.
  • Figure 10 shows the result of down-regulation of PARP in the ALS fruit fly model in Example 10, which can attenuate the neurodegeneration caused by TDP-43.
  • Example 1 The level of PARylation affects the dynamic process of assembly-disassembly of stress particles containing hnRNP A1 protein
  • the expression plasmid used in this example is pCAG-hnRNP A1-Flag, and the plasmid construction steps are as follows: human hnRNP A1 (the protein coding sequence is shown in SEQ ID NO. 1 in Table 1) from HeLa cells by PCR The cDNA (Gene ID: 3178) of ClonExpressTM II One Step Cloning Kit (Vazyme) was inserted into the pCAG plasmid by homologous recombination (this plasmid was constructed by Chen et al., for details, see Chen, Y., Wang, Y .,Erturk,A.,Kallop,D.,Jiang,Z.,Weimer,RM,Kaminker,J.,and Sheng,M.(2014).
  • activity-induced Nr4a1 regulates spinedensity and distributionpattern of excitatory synapses in pyramidal Neurons.Neuron 83,431-443), the insertion sites are EcoRI
  • the upstream primer of hnRNP A1 forward primer, F end primer:
  • hnRNP A1 downstream primer reverse primer, R end primer
  • HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (sigma) containing 10% fetal bovine serum (FBS) (Biowest). The cell growth conditions are 37°C, 5% carbon dioxide.
  • the cells were purchased from American Type Culture Collection (ATCC). Unless otherwise specified, if all the cells in the present invention are used for immunofluorescence detection experiments, they should be seeded on a 24-well plate (Corning) containing slides (Thermo) at a density of 2 ⁇ 10 5 /ml 12-18 hours in advance. Medium is used for subsequent experiments; and if used for WB detection experiments, the cells will be seeded in 12-well or 6-well plates at a density of 5 ⁇ 10 5 /ml 12-18 hours in advance for subsequent experiments.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • Plasmid transfection Transfect the plasmid pCAG-hnRNP A1-Flag constructed in 1.1.1 with PolyJet TM reagent (SignaGen) (the amount of transfection plasmid is 0.8 ⁇ g/ml, and the ratio of transfection reagent to transfection plasmid is 1 : 2 (mass volume percentage, for example, 1 microgram: 2 microliters)).
  • Cells are planted 12-18 hours in advance, and transfection starts when the cell plating rate reaches about 70%. About 6 hours after transfection, the cells were exchanged to ensure their growth status. After the foreign protein is expressed for 24-48 hours, the next step of the experiment can be performed.
  • Drug treatment In order to inhibit the level of intracellular PARylation, 20 ⁇ M PARP inhibitor Olaparib (Selleck) was added 3 hours before the plasmid-transfected samples were collected, and the control group was treated with an equal volume of DMSO.
  • RNA interference In order to increase the level of intracellular PARylation, Lipofectamine TM RNAiMAX Transfection Reagent (Invitrogen) was used to transfect small interfering RNA (Genepharma) while transfecting the above plasmids, and the subsequent sample collection was performed after 23-48 hours incubation.
  • the small interfering RNA (siRNA) sequence used in the experiment is as follows:
  • si-Ctrl 5’-UUCUCCGAACGUGUCACGUTT-3’ (SEQ ID NO.5)
  • si-hPARG 5’-GCGGUGAAGUUAGAUUACATT-3’ (SEQ ID NO.6)
  • the HeLa cells collected in 1.1.3 were treated with 100 ⁇ M sodium arsenite (Sigma) for 0, 10, and 30 minutes, respectively.
  • the HeLa cells collected in 1.1.3 were first stimulated with 100 ⁇ M sodium arsenite for 30 minutes, washed away with PBS (Sangon Biotech), and incubated with fresh medium for 0, 30, and 60 minutes.
  • Protein extraction In order to obtain the total intracellular protein obtained after the treatment in 1.1.4 above, the cells after the corresponding treatment are lysed with 2% (w/v, the same below) SDS cell lysate (the composition of the cell lysate is as follows: 50mM Tris pH 6.8, 2% SDS, 1% (w/v) ⁇ -mercaptoethanol, 12.5% (w/v) glycerol, 0.04% (w/v) bromophenol blue, protease inhibitor (Roche), 20 ⁇ M Olaparib (Selleck) And 8 ⁇ M ADP-HPD (Millopore)). After the lysed product is centrifuged at a high speed at 4°C, the supernatant is taken and added to 4 ⁇ LDS sample buffer (Invitrogen), and boiled at 95°C.
  • SDS cell lysate the composition of the cell lysate is as follows: 50mM Tris pH 6.8, 2% SDS, 1% (w/v) ⁇ -mercap
  • the membrane that has been incubated with the primary antibody is then incubated with the corresponding HRP-conjugated secondary antibody (goat anti-mouse (Sigma), goat anti-rabbit (sigma)), and then used ECL luminescent solution in the Amersham Imager 600 (GE Healthcare) machine ( Tanon) detects the corresponding protein signal.
  • HRP-conjugated secondary antibody goat anti-mouse (Sigma), goat anti-rabbit (sigma)
  • ECL luminescent solution in the Amersham Imager 600 (GE Healthcare) machine Tanon detects the corresponding protein signal.
  • (A) is a representative image of immunofluorescence of a single HeLa cell in the experiment.
  • the signal of the first channel (red) represents the TIAR protein, which serves as a marker of stress particles;
  • the signal of the second channel represents the exogenously expressed hnRNP A1 protein.
  • the stress particles in the cells will gradually appear with the stimulation of sodium arsenite; at the same time, the hnRNP A1 protein will also be transported from the nucleus to the cytoplasm and form and stress Particles with co-localized red signal.
  • (DF) Decrease the expression level of PARG enzyme in cells and delay the disassembly process of stress particles:
  • D Remove the sodium arsenite stimulation from the cells (withdraw the drug and replace with normal cell culture medium), control group (si-Ctrl) )
  • the stress particles in the cells will gradually disappear with the elution time; the dispersion and disappearance of the stress particles and the stress particles containing hnRNP A1 in the cells of the experimental group (si-hPARG, adding a siRNA that knocks down PARG) obviously lags behind.
  • the expression plasmid used in this example is pCAG-TDP-43-HA, and the plasmid construction steps are as follows: human TDP-43 (the protein coding sequence is shown in SEQ ID NO. 2 in Table 1) The cDNA of HeLa cells (Gene ID: 23435) was picked up and inserted into the pCAG plasmid by homologous recombination with the help of ClonExpressTM II One Step Cloning Kit (Vazyme). The insertion sites were EcoRI and XhoI. The HA tag is added to the primer, the primer sequence is as follows:
  • the primary antibodies used in immunofluorescence are Rabbit anti-TIAR (CST) and Mouse anti-HA (Proteintech).
  • the secondary antibody is goat anti-mouse-Alexa Fluor 488 (Thermo), goat anti-rabbit-Alexa Fluor 568 (Thermo).
  • (A) is a representative image of immunofluorescence of a single HeLa cell in the experiment.
  • the basic logic of this example is the same as that shown in Figure 1(A), except that the detection object is changed to another important ALS-related RNA binding protein TDP-43.
  • the first channel (red) signal in the figure represents the TIAR protein, which is a stress particle
  • the second channel (green) signal represents the foreign expression of TDP-43 protein.
  • the results showed that pretreatment with Olaparib, an inhibitor of PARP, delayed the formation of stress granules and TDP-43-containing stress granules in cells.
  • the arrow in the figure indicates the TDP-43 particles co-localized with the stress particles.
  • Example 3 hnRNP A1 protein can not only undergo PARylation, but also bind to PAR
  • the expression plasmid used in this example includes pCAG-hnRNP A1-K298A-Flag and pCAG-hnRNP A1-PBM mut -Flag plasmid (PBM is the abbreviation of PAR-binding motif) is the pCAG-hnRNP A1 in Example 1.
  • -Flag is a template, obtained by means of single point mutation PCR with Fast Mutagenesis Kit II (Vazyme), and the primer sequences used are as follows.
  • the cells after the corresponding treatment as described in 3.1.2 are lysed by adding 2% SDS cell lysate (the composition of the cell lysate is as follows: 50mM Tris pH 6.8, 2% SDS, 1% ⁇ -mercaptoethanol, 12.5% glycerol, 0.04% bromine Phenol blue, protease inhibitor (Roche), 20 ⁇ M Olaparib (Selleck) and 8 ⁇ M ADP-HPD (Millopore)).
  • SDS cell lysate the composition of the cell lysate is as follows: 50mM Tris pH 6.8, 2% SDS, 1% ⁇ -mercaptoethanol, 12.5% glycerol, 0.04% bromine Phenol blue, protease inhibitor (Roche), 20 ⁇ M Olaparib (Selleck) and 8 ⁇ M ADP-HPD (Millopore)).
  • SDS cell lysate the composition of the cell lysate is as follows: 50mM Tris pH 6.8, 2% SDS,
  • lysate is added to HA antibody (Mouse anti-HA (Proteintech)) and incubated overnight at 4°C.
  • Protein G beads enrich the primary antibody-sample complex for 2 hours at room temperature; for Flag-tagged hnRNP A1, the lysate is added directly M2 Affinity Gel (Sigma) was incubated at 4°C.
  • 1 ⁇ purified PARG protein was added to Incubate the mixture of M2 Affinity Gel (Sigma) and cell lysate at 37°C for 1 hour. After the incubation, the product was washed 3-5 times with lysate, then added 4 ⁇ LDS sample buffer (Invitrogen), and boiled at 95°C for subsequent research.
  • the cooked protein was separated by 10% Bis-Tris SDS-PAGE (Invitrogen), transferred to a 0.22 ⁇ m PVDF membrane (Millipore), blocked with 5% skimmed milk powder at room temperature for 1 hour, and then added the corresponding antibody for immunization Blot detection.
  • the membrane that has been incubated with the primary antibody (Rabbit anti-HA (CST), Mouse anti-Flag (Sigma), anti-pan-ADP-ribose binding reagent (Millipore)) is reused with the corresponding HRP-conjugated secondary antibody (goat anti-mouse) (Sigma), goat anti-rabbit (sigma))
  • the corresponding protein signal is detected with ECL luminescent solution (Tanon) in the Amersham Imager 600 (GE Healthcare) machine.
  • hnRNP A1 in the cell Under the stimulation of hydrogen peroxide, hnRNP A1 in the cell itself has a higher level of PARylation modification, and there are more PARylation-modified proteins that interact with it. However, if PARG protein is added to the IP system for 1 h, both hnRNP A1's own PARylation level and the PARylation-modified protein that interacts with it are significantly reduced, indicating that the signal detected in WB is indeed caused by PARylation of.
  • FIG. D Schematic diagram of the functional domain of hnRNP A1 protein.
  • Human hnRNP A1 contains two RNA recognition sequences (RNA recognition motif, RRM) and a Glycin-rich domain (GRD) at the C-terminus.
  • RRM RNA recognition motif
  • GPD Glycin-rich domain
  • the specific sequence at the top of the schematic diagram is hnRNP A1 potential PARylation site Lys298 (marked in bold). In this example, this site is mutated to Ala (marked by underline), and the subsequent description is referred to as K298A;
  • the amino acid sequence of this domain is conservative (PBM consensus).
  • “h” stands for hydrophobic amino acids and "b” stands for basic amino acids. In the example, the three key amino acids (marked in bold) of the domain were mutated (marked by underline), and they were recorded as PBM mut in subsequent experiments.
  • hnRNP A1 can not only undergo poly-ADP ribosylation modification, but also interact with other proteins that undergo such modifications by binding to PAR.
  • the picture shows the percentage of cells that can normally form stress granules in (A) (B), the percentage of cells that form stress granules containing hnRNP A1 (C), and the cells that form abnormal dots in the cytoplasm Percentage (D) and statistics of the number (E) of abnormal cytoplasmic dots formed in each cell.
  • the above results indicate that the occurrence of PARylation at position K298 may be the nuclear signal of hnRNP A1, and the combination of PBM and PAR can help hnRNP A1 to be transported to stress particles under the stimulation of sodium arsenite.
  • Examples 3 and 4 indicate that the covalent PARylation modification of hnRNP A1 pathogenic protein will affect its subcellular localization, and non-covalent binding of PAR will affect the transport of hnRNP A1 protein to stress granules under cellular stress conditions.
  • the ALS disease caused by hnRNP A1 is closely related to its abnormal subcellular localization and stress response (see "Background Technology"). Therefore, this part of the data provides the cell regulation mechanism and experimental basis for improving the level of PARylation and developing PARP inhibitors for the treatment of ALS diseases.
  • the plasmid used for hnRNP A1 protein expression is pET9d-hnRNP A1 (Addgene), which is expressed in E. coli BL21 (DE3) pLysS (TranGeneBiotech). After 0.4mM IPTG (SangonBiotech) induced expression at 25°C for 15h.
  • the obtained bacteria are collected by centrifugation, and lysed by adding a lysis solution (50mM Tris-HCl at pH 7.5, 2mM DTT, 1mM PMSF, 5% glycerin, and 0.1mg/mL RNase A).
  • the lysate was centrifuged at a high speed at 4°C to take the supernatant, and transferred to a 5ml SP column in an AKTA (GE Healthcare) machine.
  • the protein on the column is eluted with a mixture (9:1) of buffer A (50mM Tris-HCl pH 7.5, 2mM DTT and 5% glycerin) and buffer B (buffer A with 1M NaCl).
  • the eluted protein was purified with Superdex 7516/600column (GE Healthcare).
  • the purified protein was dissolved in a stock buffer (50mM Tris-HCl pH 7.5, 500mM NaCl and 2mM DTT) for subsequent experiments.
  • the purified hnRNP A1 protein is mixed with a certain concentration of PAR (Trevigen) at a certain salt concentration, and the reaction system is 50mM Tris-HCl, pH 7.5, 10% (w/v)PEG 3550 (Sigma) and 2mM DTT. After the mixture was incubated at room temperature for 3 minutes, 5ul was dropped on a glass slide for observation with an SP8 confocal microscope (Leica).
  • hnRNP A1 can spontaneously form droplets through LLPS: The picture shows the case of hnRNP A1 forming droplets through phase change at a specific protein concentration and salt ion concentration. With the increase of protein concentration and the decrease of salt ion concentration, hnRNP A1 droplets The diameter and number gradually increase.
  • Example 7 hnRNP A1 can be separated from TDP-43, and PAR can promote this process
  • the plasmid pET9d-hnRNP A1 used for in vitro expression and purification was purchased from Addgene.
  • the other two plasmids, pET-28a-TDP-43 1-274 -6 ⁇ His and pET-28a-TDP-43 274-414 -6 ⁇ His pET28a plasmid source: Addgene
  • the construction steps are basically the same as those in Example 1. Insertion position
  • the points are BamHI and XhoI.
  • the primers used are as follows:
  • TDP-43 1-274 The expression plasmid is pET-28a-TDP-43 1-274 -6 ⁇ His.
  • the plasmid is expressed in BL21E.coli (DE3) (TranGeneBiotech), after 50uM IPTG (SangonBiotech) at 19°C 16h after induced expression under conditions.
  • the obtained bacteria were collected by centrifugation and lysed by adding lysis solution (50mM Tris-HCl, 500mM NaCl, pH 8.0, 10mM imidazole, 4mM ⁇ -mercaptoethanol, 1mM PMSF, and 0.1mg/mL RNase A).
  • Ni column enrichment and purification GE Healthcare
  • Ni column enriched protein was eluted with eluent (50mM Tris-HCl, 500mM NaCl, pH 8.0, 250mM imidazole and 4mM ⁇ -mercaptoethanol), and then purified with Superdex 200 1616/600column (GE Healthcare).
  • the purified protein was finally dissolved in a storage buffer (50mM Tris-HCl pH 7.5, 300mM NaCl and 2mM DTT) for subsequent experiments.
  • TDP-43 274-414 This protein was expressed in BL21 E.coli (DE3) (TranGeneBiotech), and 50uM IPTG (SangonBiotech) was added to induce expression in inclusion bodies at 37°C.
  • the collected bacteria were lysed with denatured lysate (50mM Tris-HCl, pH 8.0 and 6M guanidine hydrochloride). The lysate was centrifuged at high speed at 4°C for 1h, and the supernatant was taken out, and then purified by Ni column.
  • the protein was eluted with denaturing elution solution (50mM Tris-HCl at pH 8.0, 6M guanidine hydrochloride and 50mM imidazole), and then purified by HPLC (Agilent), and the product obtained was lyophilized in a freeze dryer (Thermo Fisher) for use .
  • denaturing elution solution 50mM Tris-HCl at pH 8.0, 6M guanidine hydrochloride and 50mM imidazole
  • TDP-43 1-274 and hnRNP A1 proteins were diluted into the desalting reaction solution (50mM Tris-HCl, pH 7.5, 500mM NaCl and 4mM Tris(2-Carboxyethyl) Phosphine (TCEP) (Invitrogen)), DTT was removed with a desalting column (GE Healthcare). After desalting, TDP-43 1-274 and hnRNP A1 proteins were added to 5 times the volume of AlexaFluor-555 C2-malemide (Invitrogen) or AlexaFluor-647 C2-malemide (Invitrogen) and incubated for 2 hours at room temperature. The labeled protein was purified with Superdex 75 16/600 column (GE Healthcare) for use.
  • the purified hnRNP A1 and TDP-43 are mixed with the corresponding protein concentration under a certain salt concentration, and the reaction system is 50mM Tris-HCl pH 7.5, and 2mM DTT. After the mixture was incubated at room temperature for 3 minutes, 5 ⁇ l was dropped on a glass slide for observation with an SP8 confocal microscope (Leica).
  • the first channel (red) represents hnRNP A1
  • the second channel (green) represents TDP-43 1-274 .
  • the droplets can only be formed after the two are mixed, and the two droplets can be well co-located. The above results show that the droplets formed after the mixing of hnRNP A1 and TDP-43 1-274 are the result of a common phase change, rather than a separate promotion by one party to the other.
  • (G)PAR can promote the co-phase separation of hnRNP A1 and TDP-43 1-274 , and the degree of promotion is positively correlated with PAR concentration.
  • Examples 6 and 7 show that the addition of PAR can promote the co-phase separation of hnRNP A1 and TDP-43, and the co-phase separation of proteins in vitro is the molecular mechanism that mediates the formation of intracellular stress particles (see “Background Technology” for details).
  • This in vitro experiment result and the cell experiment result of Example 5 are mutually confirmed, indicating that increasing the level of PARylation will promote the interaction between hnRNP A1 and TDP-43, and strengthen the assembly of stress particles.
  • PARP inhibitors maintain the maintenance by reducing the interaction between proteins. The dynamics of stress granules and the molecular mechanism that prevent them from developing into insoluble protein aggregates that appear in ALS disease.
  • Example 8 Overexpression of hnRNP A1 or TDP-43 in motor neuron-like NSC-34 cells can cause cells toxicity
  • 293T cells were cultured in DMEM (sigma) containing 10% FBS (Biowest).
  • NSC-34 cells were cultured with RPMI 1640 medium (Gibco, 11875-093) containing 10% FBS.
  • the cell growth conditions are 37°C, 5% carbon dioxide. All cells were purchased by ATCC.
  • TUNEL staining is done with the help of TMR red in situ Cell Death Detection Kit (Sigma-Aldrich), and the specific steps are performed according to the instructions in the kit.
  • the lentivirus packaging plasmid and the corresponding exogenous hnRNP A1 or TDP-43 expression plasmid were simultaneously transfected in 293T cells.
  • the cell culture medium was collected by filtration with a 0.45 ⁇ m syringe filter (Millipore) and concentrated with Lenti-X TM Concentrator (Clontech). The concentrated product was used for subsequent NSC-34 cell infection.
  • NSC-34 cells were planted in 96-well plates (Corning) one day in advance, and the corresponding lentivirus was added. 48-72h after infection, use Cell Counting Kit-8 (CCK-8) (Dojindo) kit to determine cell viability. Simply put, add 10 ⁇ L of CCK-8 solution to each well. After incubating at 37°C for 2.5 hours, use Synergy2 microplate reader (BioTek Instruments) to detect the light absorption at 450 nm, and calculate the cell viability value according to the instructions.
  • CCK-8 Cell Counting Kit-8
  • hnRNP A1 causes NSC-34 cytotoxicity: bright field microscopy observes the abnormal morphology of NSC-34 cells overexpressing hnRNP A1, and obvious cell death occurs (A).
  • CCK-8 detects hnRNP A1 overexpression Causes a decrease in cell viability, and as the amount of lenti virus used to overexpress hnRNP A1 increases, cell viability further decreases (B).
  • TDP-43 Overexpression of TDP-43 causes NSC-34 cytotoxicity: Overexpression of TDP-43 causes abnormal morphology of NSC-34 cells (C) and a dose-dependent decrease in cell viability (D). TUNEL staining showed that overexpression of TDP-43 can cause cell death (F).
  • the first channel in the figure represents (green) TDP-43; the second channel (red) is the TUNEL signal, indicating that the cell has died; the third channel (blue) is DAPI, indicating the nucleus.
  • Example 9 The level of PARylation can regulate the cytotoxicity caused by hnRNP A1 and TDP-43
  • siRNA transfection procedure is the same as in Example 1, and the transfection time is 48-60h.
  • the corresponding siRNA sequence is as follows:
  • si-mPARG 5’-GCAGUUUCUUACACCUAUATT-3’ (SEQ ID NO.17)
  • si-mPARP1 5’-CGACGCUUAUUACUGUACUTT-3’ (SEQ ID NO.18)
  • Upstream primer 5’-AGCCTCTGACACGCTTACAC-3’; (SEQ ID NO.19)
  • Downstream primer 5’-CAGTCACACCACCTCCAACA-3’ (SEQ ID NO.20)
  • Upstream primer 5’-CACCATCTTCCAGGAGCGAG-3’; (SEQ ID NO.21)
  • Downstream primer 5’-CCTTCTCCATGGTGGTGAAGAC-3’ (SEQ ID NO.22)
  • A-C Knockdown of PARP1 can alleviate the cytotoxicity caused by hnRNP A1 or TDP-43. Infection of hnRNP A1 (A) or TDP-43 (B) in NSC-34 cells will result in a significant decrease in cell viability, and this decrease will be alleviated after knocking down PARP1 (si-PARP1).
  • C The picture shows the use of WB experiments to detect the effect of knocking down the expression level of PARP1 protein with siRNA and the situation of reducing the level of PARylaiton in cells. GAPDH was used as an internal control to prove that the protein loading amount of the two samples was the same.
  • the PARP inhibitor Olaparib can alleviate the cytotoxicity caused by TDP-43.
  • 5 ⁇ M Olaparib can reduce the cytotoxicity caused by TDP-43 overexpression. This result indicates that reducing the level of intracellular PARylation at the pharmacological level can alleviate the cytotoxicity caused by TDP-43.
  • a major feature of ALS disease is the death and loss of motor neurons.
  • the present invention uses a motor neuron-like cell NSC-34 as a model to study the effect of cytotoxicity caused by hnRNP A1 and TDP-43 on motor neurons.
  • Overexpression of hnRNP A1 or TDP-43 in NSC-34 cells can trigger abnormal effects of these two pathogenic proteins on cells in similar ALS diseases, such as changes in cell morphology and DNA breakage during apoptosis (through TdT -mediated dUTP Nick-End Labeling (TUNEL) staining method) and changes in cell number and viability (detected by Cell Counting Kit-8 (CCK-8) method), etc.
  • TUNEL TdT -mediated dUTP Nick-End Labeling
  • hnRNP A1 or TDP-43 protein will produce cytotoxicity.
  • ALS disease it means that the abnormality of these two proteins may cause the death of motor neurons and ultimately lead to the occurrence of ALS disease.
  • the results of Examples 8 and 9 show that whether the level or activity of PARP is reduced by genetic or pharmacological methods, it can effectively inhibit the motor neuron-like NSC-34 cells caused by the overexpression of hnRNP A1 and TDP-43 The toxicity. This result can be the most direct and powerful suggestion that PARP inhibitors are expected to delay motor neuron death in ALS patients.
  • the fruit flies used in the present invention are from Bloomington Drosophila Stock Center (BDSC): RNAi-Parp (#57265), elavGS (#43642), RNAi-mCherry (#35785, the RNAi-ctrl fruit flies used in the experiment), GMR-GAL4 ( #79573).
  • BDSC Bloomington Drosophila Stock Center
  • RNAi-Parp #57265)
  • elavGS #43642
  • RNAi-mCherry #35785, the RNAi-ctrl fruit flies used in the experiment
  • GMR-GAL4 #79573
  • UAS-TDP-43 Drosophila has been inserted into Drosophila genome with the help of ⁇ C31 transposase.
  • Corresponding driver fruit flies mate with specific RNAi or UAS fruit flies to produce offspring that meet the requirements. All experimental fruit flies were reared in standard corn meal
  • Drosophila protein For the extraction of Drosophila protein, the heads of the corresponding genotypes of Drosophila are separated, and RIPA lysis buffer (50mM Tris pH 8.0, 150mM NaCl, 1%NP-40, 5mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS is added , Protease inhibitors) and fully grind on ice. After the lysed product is centrifuged at four degrees of high speed, take the supernatant and add 4x LDS sample buffer (Invitrogen), and boil at 95°C.
  • RIPA lysis buffer 50mM Tris pH 8.0, 150mM NaCl, 1%NP-40, 5mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS is added , Protease inhibitors
  • the pellet obtained by centrifugation after lysis of RIPA is lysed with a 9M urea buffer (9M urea, 50mM Tris buffer, pH 8.0) equal to the soluble volume, and 4x LDS sample buffer (Invitrogen) is also added, 95°C Boil for follow-up research.
  • 9M urea buffer 9M urea, 50mM Tris buffer, pH 8.0
  • 4x LDS sample buffer Invitrogen
  • Drosophila mRNA For the extraction of Drosophila mRNA, the heads of the corresponding genotypes of Drosophila are separated, after adding Trizol (Invitrogen), thoroughly grinding with a grinding pestle, adding chloroform extraction, isopropanol precipitation to obtain mRNA, adding DNase (Promega) Eliminate contamination of genomic DNA. Take 1 ⁇ g of mRNA for reverse transcription using High-Capacity cDNA Reverse Transcription Kit (Applied biosystems). The obtained cDNA is mixed with SYBR Green qPCR Master Mix (Bimake) and target gene primers in a certain proportion, and then subjected to qPCR detection (QuantStudio TM 6 Flex Real-Time PCR system (Life Technologies)). All primers are as follows:
  • Upstream primer 5'-ATGAAGTACGGAGGCCAACC-3' (SEQ ID NO.23);
  • Downstream primer 5’-TCTTCACCTGACGCAAACCA-3’ (SEQ ID NO.24)
  • Upstream primer 5'-GAGCGCGGTTACTCTTTCAC-3' (SEQ ID NO.25);
  • Drosophila crawling ability test On the first day of fruit flies emergence, pick out flies of the corresponding genotype, and raise each group of 20 in a diet containing 80 ⁇ g/ml RU486 (TCI) to induce transgene expression. On the specified date, divide 20 fruit flies into 5-8 transparent plastic tubes, shake the plastic tube gently, shake the fruit flies to the bottom of the tube, start timing, and record that they can climb 3cm in height within 10s Calculate the percentage of the number of fruit flies (the whole fruit flies in each tube are used as an independent sample to participate in the follow-up statistics, the same below). All experiments were repeated three times.
  • Drosophila viability test On the first day of fruit fly emergence, select the corresponding genotype of fruit flies and divide them into groups of 20 into 7-9 tubes, and raise them in food containing 80 ⁇ g/ml RU486 (TCI). All fruit flies used for viability testing are replaced with fresh food added every three days, the number of fruit flies that die in each tube is recorded each time, and the survival rate of fruit flies is calculated (flies that fly away accidentally are not counted in the total) .
  • TCI 80 ⁇ g/ml RU486
  • A-E Knockdown of PARP in Drosophila can alleviate the optic nerve degeneration caused by the TDP-43 transgene.
  • A The picture shows the external eye of a single representative fruit fly in the experiment. Use the GMR driver to express in the photoreceptor nerve of Drosophila (mating Drosophila strains according to the listed genotypes, and the transgenic sequence can be automatically expressed in the specific cells of the offspring Drosophila.
  • RNAi-Parp Simultaneous expression of RNAi-Parp in this transgenic flies (same as above, mating fruit flies according to the listed genotypes, the transgenic sequence can be automatically expressed in specific cells of the offspring fruit flies) can well alleviate the TDP -43 caused by neurodegenerative disease.
  • the picture in the upper left corner is an enlargement of the area in the white box.
  • C qPCR detection of mRNA level of Parp in the head of RNAi-Parp transgenic Drosophila.
  • Example 10 show that not only in vitro and cell experiments, but also at the level of ALS in vivo animal models and the overall level of living organisms, reducing the level of PARP enzyme is effective in inhibiting the degeneration of neuronal morphology and function caused by excessive TDP-43. Even the sharp shortening of life span has obvious improvement effects, suggesting that the development of PARP inhibitors to treat ALS disease has a good prospect.
  • the PARP inhibitor Olaparib in the present invention is a clinical drug approved by the FDA for the treatment of breast cancer, ovarian cancer, prostate cancer and other tumors (Fong et al., 2009; Tutt et al., 2010), and its toxic and side effects have been clinically tested. Argument.
  • the present invention shows that the inhibitor has a significant inhibitory effect on neurocytotoxicity induced by hnRNP A1 and TDP-43. Therefore, the inhibitor is more likely to be used in the development of ALS clinical drugs and has less risk.

Abstract

一种治疗神经退行性疾病或RNA结合蛋白发生异常导致的疾病的药物组合物及其应用，特别是在治疗ALS中的应用。药物组合物能够明显增强含有RNA结合蛋白如hnRNP A1和TDP-43蛋白的应激颗粒的动态性；影响RNA结合蛋白与其它多聚ADP核糖基化修饰蛋白或其它PAR结合蛋白之间的相互作用；影响RNA结合蛋白的亚细胞定位和应激响应；影响RNA结合蛋白的液-液相分离和聚集倾向；影响RNA结合蛋白之间的共相分离；影响RNA结合蛋白在细胞中的相互作用；对RNA结合蛋白引起的神经细胞毒性发挥了显著的抑制作用。

Description

治疗神经退行性疾病或RNA结合蛋白发生异常导致的疾病的药物组合物及其应用 技术领域
本发明涉及一种治疗神经退行性疾病或RNA结合蛋白发生异常导致的疾病的药物组合物及其应用。
背景技术
1.肌萎缩性脊髓侧索硬化症(Amyotrophic lateral sclerosis,ALS)
ALS疾病概述：ALS是一种运动神经元退化、变性导致的神经系统疾病，平均每100,000人中有1-2人发病(Bento-Abreu et al.,2010)。ALS疾病主要表现为下行运动神经元(包括脑干及腹角)和上行运动神经元(包括皮质等)的大量丢失，主要的症状包括：肌肉萎缩，肌肉痉挛，构音障碍，吞咽困难，在后期患者丧失基本的运动能力，最后会由于呼吸衰竭而死亡(Mitchell and Borasio,2007；Nguyen et al.,2018；van Es et al.,2017)。
ALS治疗和药物研发现状：ALS疾病现在仍属于不治之症。目前被美国食品药物管理局(FDA)批准的临床用药仅有Riluzole和Edaravone。Riluzole通过抑制谷氨酸能的活性来起到延缓ALS疾病进程的作用，但是该药特异性较差，仅仅能延长病人3-6个月的生命，并且伴有恶心，晕眩甚至发生肺炎等的副作用(Lacomblez et al.,1996；Tripathi and Al-Chalabi,2008)。Edaravone是一种自由基清除剂，三期临床的结果显示Edaravone能够一定程度上缓解ALS病人的身体机能损伤，并在2017年该药被FDA批准用于ALS的疾病治疗，但该药只对于符合post-hoc分析标准的小部分病人有效(Abe et al.,2017)。
已知的ALS致病机理：ALS致病机理较为复杂，具体分子机制尚不明确，其中比较常见的是由于RNA结合蛋白发生基因突变导致的ALS。在正常细胞里，RNA结合蛋白通过与RNA结合形成核糖核蛋白(ribonucleoprotein,RNP)复合物发挥功能。在细胞应激的情况下，RNP复合物会形成应激颗粒(stress granules)维持细胞稳态(Buchan et al.,2009)。编码这些蛋白的基因发生突变时，可以影响RNP颗粒的形成和功能、促使其异常聚集，而这种异常聚集是包括ALS在内的多种神经退行性疾病的重要病理标志。细胞中RNA结合蛋白有数百种，目前已知与ALS疾病相关的RNA结合蛋白主要包括heterogeneous nuclear ribonucleoprotein A1(hnRNP A1)、Transactivation response element DNA-binding protein 43(TDP-43)、Fused in sarcoma(FUS)、TATA-binding protein-associated factor 15(TAF15)、Ewing sarcoma breakpoint region 1(EWSR1)、T-cell intracytoplasmic antigen 1(TIA-1)等(Harrison and Shorter,2017)。
ALS主要致病蛋白hnRNP A1和TDP-43：hnRNP A1与TDP-43在正常生理状态下 主要定位在细胞核内，参与调控多种细胞生理功能。关于它们导致ALS疾病的分子机制，目前已知的包括：(1)hnRNP A1和TDP-43蛋白的异常翻译后修饰，例如过度磷酸化、泛素化等和细胞内异常聚集(Neumann et al.,2006)。(2)在ALS病人组织中，hnRNP A1和TDP-43蛋白均被发现有异常定位在细胞胞浆中、并形成病理性的包涵体的现象(Kim et al.,2013；Neumann et al.,2006)。(3)hnRNP A1和TDP-43所参与的应激颗粒的形成和调控发生异常：hnRNP A1和TDP-43蛋白都是细胞应激颗粒的重要组成成分；hnRNP A1和TDP-43的基因突变可以影响它们定位到应激颗粒并与之结合的能力；相应的，调节应激颗粒的形成能够缓解由于这些RNA结合蛋白引起的神经毒性(Elden et al.,2010；Liu-Yesucevitz et al.,2010；Li et al.,2013；Kim et al.,2013；Kim et al.,2014；Naruse et al.,2018)。此外，hnRNP A1和TDP-43导致ALS发生的分子机制还与其调控RNA处理和加工的功能异常以及对线粒体的异常影响有关。
LLPS与ALS的关系：hnRNP A1等RNA结合蛋白能够与RNA相互作用形成RNP颗粒，特别是形成细胞应激颗粒。最新的研究显示，这些RNA结合蛋白在体外实验中能够通过液-液相分离(Liquid-Liquid Phase separation,LLPS)形成与细胞中应激颗粒类似的液滴(Lin et al.,2015)。这些液滴具有高度的流动性(dynamics)和可逆性(reversible)，能与周围的其他液滴进行融合或分离。当外界环境条件改变时，经由LLPS形成的液滴不断固化、逐渐形成淀粉样聚集(Hyman et al.,2014；Lin et al.,2015；Molliex et al.,2015；Wang et al.,2014)。这一过程与体内应激颗粒在ALS疾病进程中逐渐丢失流动性、可逆性，最终发展成不可逆的病理性蛋白包涵体的过程类似，因此可以用来表征RNA结合蛋白发生聚集的倾向性。
2.多聚ADP核糖基化修饰(poly(ADP-ribosyl)ation,PARylation)
PARylation概述：蛋白PARylation修饰这一可逆反应由聚合酶poly(ADP-ribose)polymerase(PARP)家族和水解酶poly(ADP-ribose)glycohydrolase(PARG)催化。PARylation修饰参与调控的细胞功能主要有：(1)参与调节染色质结构以及基因转录；(2)协助DNA损伤修复复合物的组装，促进其发挥功能；(3)激活蛋白酶体清除损伤的组蛋白；(4)推动蛋白向Cajal body中转运；(5)影响蛋白的核内外转运；(6)促进线粒体释放凋亡诱导因子(apoptosis inducing factor,AIF)及其向核内的运输；(7)参与调节应激颗粒的组装和正常的功能(Luo and Kraus,2012)。
PARP抑制剂的临床应用和药物开发：PARP抑制剂的临床应用主要集中在肿瘤治疗方面，如PARP1抑制剂Olaparib被FDA批准用于与BRCA1和BRCA2基因突变相关的卵巢癌、乳腺癌、前列腺癌等癌症的治疗(Fong et al.,2009；Tutt et al.,2010)。此外，少 量实验室和临床前研究提示降低PARylation水平对于亨廷顿式舞蹈症、大脑局部缺血和轴突损伤等情况也具有一定神经保护作用(Brochier et al.,2015；Cardinale et al.,2015；Egi et al.,2011；Teng et al.,2016)。但目前尚无任何相关的研究或报道称，可以将PARP抑制剂例如Olaparib用于ALS等疾病的治疗中。
3.PARylation与ALS疾病
目前关于PARylation在ALS中的作用在此之前几乎没有报道，仅在近期的一篇文献中有提及细胞中游离的多聚ADP核糖(poly(ADP-ribose),PAR)可能与TDP-43结合并影响其定位到应激颗粒(McGurk et al.,2018)。但是McGurk等人只研究了非共价性PAR结合(PAR-binding)对TDP-43这单独一个致病蛋白的影响，并且只观察到TDP-43与PAR链的结合会影响其与细胞应激颗粒的定位；McGurk等人认为Tankyrase(即PARP5)是催化TDP-43PARylation修饰的主要酶；Tankyrase主要定位在细胞胞浆内。McGurk等人的研究中所使用的Tankyrase抑制剂XAV939和G007-LK均非FDA批准的临床用药，仅为实验室研究用抑制剂；且其研究中并未对XAV939和G007-LK用于抑制TDP-43引发对神经毒性进行有效性和安全性的实验。
发明内容
本发明所要解决的技术问题是为了克服现有技术中缺乏有效治疗神经退行性疾病或RNA结合蛋白发生异常导致的疾病的药物的缺陷，提供一种治疗神经退行性疾病或RNA结合蛋白发生异常导致的疾病的药物组合物及其应用，特别是在治疗肌萎缩性脊髓侧索硬化症(ALS)疾病中的应用。发明人经研究发现，本发明所述药物组合物如Olaparib能够明显增强含有RNA结合蛋白如hnRNP A1(heterogeneous nuclear ribonucleoprotein A1)和TDP-43(Transactivation response element DNA-binding protein 43)蛋白的应激颗粒的动态性，主要是抑制应激颗粒的组装和/或促进应激颗粒的去组装；影响RNA结合蛋白如hnRNP A1和其它多聚ADP核糖基化修饰的蛋白之间的相互作用；影响RNA结合蛋白如hnRNP A1和其它PAR结合蛋白之间的相互作用；影响RNA结合蛋白如hnRNP A1的亚细胞定位和应激响应；影响RNA结合蛋白如hnRNP A1的液-液相分离和聚集倾向；影响RNA结合蛋白如hnRNP A1和TDP-43蛋白的共相分离；影响RNA结合蛋白如hnRNP A1和TDP-43蛋白在细胞中的相互作用。此外，在类运动神经元NSC-34细胞中所述药物组合物如Olaparib对RNA结合蛋白如hnRNP A1和TDP-43引起的神经细胞毒性发挥了显著的抑制作用。
目前，PARP抑制剂多用于肿瘤治疗方面，并且无任何研究或报道显示降低细胞内多聚ADP核糖基化修饰水平的药物如Olaparib可用于ALS等神经退行性疾病的治疗中。 而本发明首次对一种FDA批准的用于肿瘤治疗的PARP小分子抑制剂Olaparib进行了ALS疾病治疗的相关实验，并且发现该抑制剂对于hnRNP A1和TDP-43引发的神经细胞毒性均有明显抑制作用，故该抑制剂可用于开发治疗ALS等相关疾病的临床用药。
为了解决上述技术问题，本发明提供了一种治疗神经退行性疾病的药物组合物，所述药物组合物包括降低细胞内多聚ADP核糖基化修饰水平的药物，优选降低细胞内RNA结合蛋白的多聚ADP核糖基化修饰水平的药物。
较佳地，所述RNA结合蛋白为hnRNP A1和/或TDP-43。
较佳地，所述神经退行性疾病为肌萎缩性脊髓侧索硬化症(ALS)。
为了解决上述技术问题，本发明提供了一种治疗RNA结合蛋白发生异常导致的疾病的药物组合物，所述药物组合物包括降低细胞内多聚ADP核糖基化修饰水平的药物，优选降低细胞内所述RNA结合蛋白的多聚ADP核糖基化修饰水平的药物；所述RNA结合蛋白优选为hnRNP A1和/或TDP-43。
较佳地，所述RNA结合蛋白发生异常导致的疾病为肌萎缩性脊髓侧索硬化症(ALS)。
较佳地，所述治疗RNA结合蛋白发生异常导致的疾病包括以下一种或多种：
(a)RNA结合蛋白的异常翻译后修饰引起的细胞内异常聚集导致的疾病；
(b)RNA结合蛋白的异常亚细胞定位导致的疾病；
(c)RNA结合蛋白所参与的应激颗粒的形成和/或调控发生异常导致的疾病。
较佳地，所述治疗RNA结合蛋白发生异常导致的疾病包括以下一种或多种：
(a)RNA结合蛋白结合和/或调节RNA的加工、剪切、转运和/或翻译发生异常导致的疾病；
(b)RNA结合蛋白如hnRNP A1发生共价的多聚ADP核糖基化修饰影响所述RNA结合蛋白如hnRNP A1与其它蛋白如TDP-43蛋白的相互作用和/或亚细胞定位导致的疾病；
(c)RNA结合蛋白如hnRNP A1通过非共价结合PAR影响所述RNA结合蛋白如hnRNP A1与其它蛋白如TDP-43蛋白的相互作用导致的疾病；
(d)RNA结合蛋白通过非共价结合PAR影响其在细胞应激条件下向应激颗粒的转运导致的疾病；
(e)RNA结合蛋白通过非共价结合PAR影响其在细胞中的可溶性改变从而引起细胞稳态破坏导致的疾病。
较佳地，本发明中所述降低细胞内多聚ADP核糖基化修饰水平的药物为能够提高PARG水解酶表达水平和/或降低PARP聚合酶表达水平的药物；
更佳地，所述能够提高PARG水解酶表达水平的药物为PARG水解酶激动剂；
更佳地，所述能够降低PARP聚合酶表达水平的药物为PARP聚合酶抑制剂；所述PARP聚合酶抑制剂优选Olaparib。
为了解决上述技术问题，本发明提供了一种治疗上述神经退行性疾病或上述RNA结合蛋白发生异常导致的疾病的方法，所述方法包括利用上述药物组合物进行治疗。
较佳地，所述药物组合物通过以下的一种或多种机制进行治疗：
(a)影响hnRNP A1和/或TDP-43形成应激颗粒，优选抑制应激颗粒的组装和/或促进应激颗粒的去组装；
(b)影响hnRNP A1和其它多聚ADP核糖基化修饰的蛋白之间的相互作用；
(c)影响hnRNP A1和其它PAR结合蛋白之间的相互作用；
(d)影响hnRNP A1的亚细胞定位和/或应激响应；
(e)影响hnRNP A1的液-液相分离和/或聚集倾向；
(f)影响hnRNP A1和TDP-43蛋白的共相分离；
(g)影响hnRNP A1和TDP-43蛋白在细胞中的相互作用；
(h)抑制hnRNP A1和/或TDP-43引起的神经细胞毒性。
较佳地，所述药物组合物最终可以抑制hnRNP A1和/或TDP-43所引起的神经变性。所述神经变性较佳地主要表现为：1)在类运动神经元NSC-34细胞中的毒性抑制作用，和/或，2)对ALS疾病果蝇模型中神经元退化、运动能力降低和寿命缩短等表型的缓解作用。
为了解决上述技术问题，本发明提供了上述药物组合物在治疗上述神经退行性疾病或上述RNA结合蛋白发生异常导致的疾病中的应用。
为了解决上述技术问题，本发明提供了上述药物组合物在制备治疗上述神经退行性疾病或上述RNA结合蛋白发生异常导致的疾病的药物中的应用。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明的药物组合物如Olaparib能够明显增强含有RNA结合蛋白如hnRNP A1和TDP-43蛋白的应激颗粒的动态性，主要是抑制应激颗粒的组装和/或促进应激颗粒的去组装；影响RNA结合蛋白如hnRNP A1和其它多聚ADP核糖基化修饰的蛋白之间的相互作用；影响RNA结合蛋白如hnRNP A1和其它PAR结合蛋白之间的相互作用；影响RNA结合蛋白如hnRNP A1的亚细胞定位和应激响应；影 响RNA结合蛋白如hnRNP A1的液-液相分离和聚集倾向；影响RNA结合蛋白如hnRNP A1和TDP-43蛋白的共相分离；影响RNA结合蛋白如hnRNP A1和TDP-43蛋白在细胞中的相互作用。此外，在类运动神经元NSC-34细胞中所述药物组合物如Olaparib对RNA结合蛋白如hnRNP A1和TDP-43引起的神经细胞毒性发挥了显著的抑制作用。
附图说明
图1显示了实施例1中PARylation水平影响含有hnRNP A1蛋白的应激颗粒的组装-去组装的动态过程结果图。
图2显示了实施例2中PARylation水平影响含有TDP-43蛋白的应激颗粒的组装-去组装的动态过程结果图。
图3显示了实施例3中hnRNP A1蛋白既能够发生PARylation，又可以与PAR结合的结果图。
图4显示了实施例4中PARylation或者PAR的结合能够影响hnRNP A1的亚细胞定位和应激响应的结果图。
图5显示了实施例5中PARylation能够调节hnRNP A1与TDP-43的相互作用的结果图。
图6显示了实施例6中PAR能够促进hnRNP A1在体外发生液-液相分离的结果图。
图7显示了实施例7中hnRNP A1能够和TDP-43发生共相分离，并且PAR能够促进这一过程的结果图。
图8显示了实施例8中在类运动神经元NSC-34细胞中过表达hnRNP A1或者TDP-43会引起细胞毒性的结果图。
图9显示了实施例9中PARylation水平能够调节由于hnRNP A1和TDP-43引起的细胞毒性的结果图。
图10显示了实施例10中在ALS果蝇模型中下调PARP可以减弱由于TDP-43引起神经退行性病变的结果图。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
表1
Figure PCTCN2019074579-appb-000001
Figure PCTCN2019074579-appb-000002
Figure PCTCN2019074579-appb-000003
实施例1.PARylation水平影响含有hnRNP A1蛋白的应激颗粒的组装-去组装的动态过程
1.1实验步骤
1.1.1质粒构建
本实施例中所用到的表达质粒为pCAG-hnRNP A1-Flag，质粒构建步骤如下：人源hnRNP A1(蛋白编码序列如表1中的SEQ ID NO.1所示)以PCR的方式从HeLa细胞的cDNA(Gene ID:3178)中钓取，借助ClonExpressTM II One Step Cloning Kit(Vazyme)以同源重组的方式插入到pCAG质粒(该质粒由陈等构建，具体参见Chen,Y.,Wang,Y.,Erturk,A.,Kallop,D.,Jiang,Z.,Weimer,R.M.,Kaminker,J.,and Sheng,M.(2014).Activity-induced Nr4a1 regulates spine density and distribution pattern of excitatory synapses in pyramidal neurons.Neuron 83,431-443)中，插入位点为EcoRI和XhoI。Flag标签加在引物中，引物序列如下：
hnRNP A1的上游引物(forward primer，F端引物)：
Figure PCTCN2019074579-appb-000004
hnRNP A1的下游引物(reverse primer，R端引物)：
Figure PCTCN2019074579-appb-000005
1.1.2细胞培养
HeLa细胞在含有10％胎牛血清(FBS)(Biowest)的Dulbecco’s modified Eagle’s  medium(DMEM)(sigma)中培养。细胞生长条件为37℃，5％二氧化碳。细胞购自American Type Culture Collection(ATCC)。除特殊说明外，本发明中的所有细胞如果用于免疫荧光检测实验的细胞，提前12-18小时以2×10 5/ml的密度种在含有玻片(Thermo)的24孔板(Corning)中以用于后续实验；而如果用于WB检测实验，细胞将提前12-18小时以5×10 5/ml的密度种在12孔或者6孔板中用于后续实验。
1.1.3细胞处理
质粒转染：将1.1.1中构建的质粒pCAG-hnRNP A1-Flag用PolyJet TMreagent(SignaGen)进行转染(转染质粒用量为0.8μg/ml，转染试剂与转染质粒用量比值为1:2(质量体积百分比，例如1微克：2微升))。细胞提前12-18小时种板，待细胞铺板率达到70％左右时开始转染。转染后6小时左右，细胞换液以保证生长状态。待外源蛋白表达24-48h后方可进行下一步实验操作。
药物处理：为了抑制细胞内PARylation水平，在收上述经质粒转染后的样品前3小时加入20μM的PARP抑制剂Olaparib(Selleck)，对照组用等体积的DMSO处理。
RNA干扰：为了提高细胞内PARylation水平，在转染上述质粒的同时用Lipofectamine TMRNAiMAX Transfection Reagent(Invitrogen)转染小干扰RNA(Genepharma)，孵育23-48小时后进行后续收样操作。实验所用到的小干扰RNA(siRNA)序列如下：
si-Ctrl：5’-UUCUCCGAACGUGUCACGUTT-3’(SEQ ID NO.5)
si-hPARG：5’-GCGGUGAAGUUAGAUUACATT-3’(SEQ ID NO.6)
1.1.4应激颗粒诱导与恢复
为了诱导应激颗粒的产生，上述1.1.3中收样后的HeLa细胞分别用100μM亚砷酸钠(Sigma)处理0、10、30分钟。
对于刺激移除实验，上述1.1.3中收样后的HeLa细胞先用100μM亚砷酸钠刺激30min后，PBS(Sangon Biotech)洗去，分别换新鲜培养基孵育0、30、60min。
1.1.5免疫荧光
将上述1.1.4中处理后所得的细胞用PBS洗一次，加入4％多聚甲醛(Sangon Biotech)固定15min，再用0.5％Triton-X100(Sigma)溶液通透10min，最后加入含有3％山羊血清(Sigma)的PBST溶液(0.1％Tween 20(Sigma)in PBS)室温封闭1小时，加入抗体(Rabbit anti-TIAR(CST),Mouse anti-Flag(Sigma))4℃过夜孵育。PBST室温洗三次，室温孵育1小时。再次用PBST室温洗三次后，加入荧光二抗(goat anti-mouse-Alexa Fluor 488(Thermo),goat anti-rabbit-Alexa Fluor 568(Thermo))进行标记，再用Vectashield Antifade  Mounting Medium with DAPI(Vector Laboratories)封片。在Leica TCS SP8共聚焦显微镜的100×油镜下观察并采集信号。
1.1.6免疫印迹(Western Blot，WB)
蛋白提取：为了得到上述1.1.4中处理后所得的细胞内总蛋白，相应处理后的细胞加入2％(w/v，下同)SDS细胞裂解液裂解(细胞裂解液成分如下：50mM Tris pH 6.8，2％SDS，1％(w/v)β-巯基乙醇，12.5％(w/v)甘油，0.04％(w/v)溴酚蓝，蛋白酶抑制剂(Roche)，20μM Olaparib(Selleck)和8μM ADP-HPD(Millopore))。裂解后的产物经过4℃高速离心后，取上清加入4×LDS sample buffer(Invitrogen)，95℃煮沸。
免疫印迹检测：上述煮过的蛋白用10％Bis-Tris SDS-PAGE(Invitrogen)分离后，转印到0.22μm PVDF膜(Millipore)，5％脱脂奶粉封闭后，分别加入相应抗体(anti-pan-ADP-ribose binding reagent(Millipore),mouse anti-Tubulin(MLB),rabbit anti-PRAG(CST),mouse anti-GAPDH(Proteintech))作免疫印迹检测。孵育过一抗的膜再用偶联HRP的相应二抗(goat anti-mouse(Sigma),goat anti-rabbit(sigma))孵育后，在Amersham Imager 600(GE Healthcare)机器中用ECL发光液(Tanon)检测相应蛋白信号。
1.2.实验结果(如图1所示)
(A-C)抑制PARP酶活性可以阻碍应急颗粒的组装：(A)图为实验中单个HeLa细胞的免疫荧光代表性图片。第一个通道(红色)信号代表TIAR蛋白，作为应激颗粒的标志物；第二个通道(绿色)信号代表外源表达的hnRNP A1蛋白。在对照组中(DMSO预处理3小时)细胞中的应激颗粒会随着亚砷酸钠的刺激逐渐出现；与此同时，hnRNP A1蛋白也会从细胞核内向包浆中转运并形成与应激颗粒红色信号共定位的颗粒。在实验组中(PARP的抑制剂Olaparib预处理3小时)，细胞中应激颗粒以及含有hnRNP A1的应激颗粒的形成被延缓。图中箭头指示与应激颗粒共定位的hnRNP A1。(B)图为对(A)图所示实验中对照组(DMSO)和实验组(Olaparib)中出现应激颗粒的细胞、以及出现含有hnRNP A1蛋白的应激颗粒的细胞比例(％)在给予亚砷酸钠刺激后不同时间点的统计。(C)图为使用Western Blot(WB)实验验证使用PARP抑制剂Olaparib可以显著降低细胞中PARylation水平。其中GAPDH(Proteintech)作为内参证明两组样品的蛋白上样量一致。这组实验结果显示，使用PARP抑制剂Olaparib降低细胞PARylation水平可以明显减缓应激颗粒的形成。
(D-F)降低细胞中PARG酶表达水平延缓应激颗粒的去组装过程：(D)将细胞中的亚砷酸钠刺激去除(撤药并替换为正常细胞培养液)，对照组(si-Ctrl)细胞中的应激颗粒会随洗脱时间逐渐消失；实验组(si-hPARG，加入敲低PARG的siRNA)的细胞中 应激颗粒和含有hnRNP A1的应激颗粒的离散和消失明显滞后。(E)图为对(D)图所示实验中对照组(si-Ctrl)和实验组(si-hPARG)中呈现应激颗粒的细胞、以及呈现含有hnRNP A1蛋白的应激颗粒的细胞的比例(％)在亚砷酸钠刺激去除后不同时间点的统计。(F)图为使用WB实验检测使用siRNA敲低hPARG蛋白表达水平的效果以及提高细胞中PARylaiton水平的情况。Tubulin(MBL International Corporation)作为内参证明两组样品的蛋白上样量一致。这组实验结果表明，降低细胞中PARG水解酶表达量从而提高细胞内PARylation水平可以显著推迟hnRNP A1参与的应激颗粒的去组装。
图1中所有实验均重复三次，每组统计大约100个细胞。统计学差异的显著性通过非配对、双侧Student’s t-test方法检验p value测定。其中，ns代表两组间无显著差异；*为p<0.05,**为p<0.01。下同。
实施例2.PARylation水平影响含有TDP-43蛋白的应激颗粒的组装-去组装的动态过程
2.1.实验步骤
2.1.1质粒构建
本实施例中所用到的表达质粒为pCAG-TDP-43-HA，质粒构建步骤如下：人源TDP-43(蛋白编码序列如表1中的SEQ ID NO.2所示)以PCR的方式从HeLa细胞的cDNA中钓取(Gene ID:23435)，借助ClonExpressTM II One Step Cloning Kit(Vazyme)以同源重组的方式插入到pCAG质粒中，插入位点为EcoRI和XhoI。HA标签加在引物中，引物序列如下：
TDP-43的上游引物:
Figure PCTCN2019074579-appb-000006
TDP-43的下游引物:
Figure PCTCN2019074579-appb-000007
2.1.2本实施例中所用到的其余实验试剂及实验步骤同实施例1。
其中，免疫荧光中所使用的一抗为Rabbit anti-TIAR(CST)，Mouse anti-HA(Proteintech)。二抗为goat anti-mouse-Alexa Fluor 488(Thermo)，goat anti-rabbit-Alexa Fluor 568(Thermo)。
2.2.实验结果(如图2所示)
(A-B)抑制PARP酶活性可以阻碍应激颗粒的组装：(A)图为实验中单个HeLa细 胞的免疫荧光代表性图片。本实施例基本逻辑同图1(A)，仅将检测对象更改为另外一种重要的ALS相关RNA结合蛋白TDP-43，图中第一个通道(红色)信号代表TIAR蛋白，为应激颗粒的标志物；第二个通道(绿色)信号代表外源表达的TDP-43蛋白。结果表明：PARP的抑制剂Olaparib预处理会使得细胞中应激颗粒以及含有TDP-43的应激颗粒的形成被延缓。图中箭头指示与应激颗粒共定位的TDP-43颗粒。(B)图为对(A)图所示实验中对照组(DMSO)和实验组(Olaparib)中出现应激颗粒的细胞、以及出现含有TDP-43蛋白的应激颗粒的细胞比例(％)在给予亚砷酸钠刺激后不同时间点的统计。这组实验结果显示，使用PARP抑制剂Olaparib降低细胞PARylation水平可以明显减缓TDP-43参与的应激颗粒的形成。
(C-D)降低细胞中PARG酶表达水平延缓应激颗粒的去组装过程：(C)该图的基本逻辑同图(1D)，结果显示使用siRNA敲低PARG导致细胞中应激颗粒和含有TDP-43的应激颗粒在刺激移除后离散和消失的速度明显减慢。(D)图为对(C)图所示实验中对照组(si-Ctrl)和实验组(si-hPARG)中呈现应激颗粒的细胞、以及呈现含有TDP-43蛋白的应激颗粒的细胞的比例(％)在亚砷酸钠刺激去除后不同时间点的统计。这组实验结果表明，降低细胞中PARG水解酶表达量从而提高细胞内PARylation水平可以显著推迟TDP-43参与的应激颗粒的去组装。
图2中所有实验均重复三次，每组统计大约100个细胞。统计学差异的测定同实施例1。
由实施例1、2的结果可知，PARylation的水平会影响hnRNP A1和TDP-43参与的应激颗粒的组装-去组装动态过程。并且由于现有技术中已有报道称应激颗粒与ALS等神经退行性疾病关系密切(Li et al.,2013)，从而说明PARylation与ALS疾病之间存在相关性。
实施例3.hnRNP A1蛋白既能够发生PARylation，又可以与PAR结合
3.1.实验步骤
3.1.1质粒构建
本实施例中所用到的表达质粒包括pCAG-hnRNP A1-K298A-Flag和pCAG-hnRNP A1-PBM mut-Flag质粒(PBM是PAR-binding motif的简称)是以实施例1中的pCAG-hnRNP A1-Flag为模板，用Fast Mutagenesis Kit II(Vazyme)以单点突变PCR的方式获得，所用的引物序列如下。
pCAG-hnRNP A1-K298A-Flag对应的引物:
hnRNP A1-K298A的上游引物：
Figure PCTCN2019074579-appb-000008
hnRNP A1-K298A的下游引物：
Figure PCTCN2019074579-appb-000009
pCAG-hnRNP A1-PBM mut-Flag对应的引物：
hnRNP A1-PBM mut的上游引物：
Figure PCTCN2019074579-appb-000010
hnRNP A1-PBM mut的下游引物：
Figure PCTCN2019074579-appb-000011
3.1.2细胞内PARylation诱导
HeLa细胞培养、质粒转染等步骤参见实施例1所述。为了提高转染3.1.1所述质粒后的HeLa细胞内PARylation的水平，在收样前将细胞培养基移走，加入500μM双氧水(稀释于PBS中)，37℃孵育10min。刺激后的HeLa细胞可用于后续的实验。
3.1.3蛋白提取
按照3.1.2所述的相应处理后的细胞加入2％SDS细胞裂解液裂解(细胞裂解液成分如下：50mM Tris pH 6.8,2％SDS,1％β-巯基乙醇,12.5％甘油，0.04％溴酚蓝,蛋白酶抑制剂(Roche),20μM Olaparib(Selleck)和8μM ADP-HPD(Millopore))。裂解后的产物经过4℃高速离心后，取上清加入4×LDS sample buffer(Invitrogen)，95℃煮沸。
3.1.4免疫沉淀(immunoprecipitation,IP)
按照3.1.2处理后的细胞用下述裂解液裂解：25mM Tris-HCl pH 7.4，150mM NaCl，1％NP-40，1mM EDTA，5％甘油，蛋白酶抑制剂(Roche)，20μM Olaparib(Selleck)和8μM ADP-HPD(Millopore)。对于带有HA标签的TDP-43的富集，裂解产物加入HA抗体(Mouse anti-HA(Proteintech))4℃过夜孵育后用
Figure PCTCN2019074579-appb-000012
Protein G beads(Novex)室温富集一抗-样品复合物2小时；对于Flag标签的hnRNP A1而言，裂解产物直接加入
Figure PCTCN2019074579-appb-000013
M2 Affinity Gel(Sigma)与4℃孵育。为了在体外消解多聚核糖基团，1μ纯化的PARG蛋白(Sigma)加入到
Figure PCTCN2019074579-appb-000014
M2 Affinity Gel(Sigma)与细胞裂解液的混合物中，37℃孵育1小时。孵育后的产物用裂解液洗3-5次后加入4×LDS sample buffer(Invitrogen)，95℃煮沸以便后续研究。
3.1.5免疫印迹(Western Blot,WB)
按照3.1.3煮过的蛋白用10％Bis-Tris SDS-PAGE(Invitrogen)分离后，转印到0.22μm PVDF膜(Millipore)上，5％脱脂奶粉室温封闭1小时，然后加入相应抗体作免疫印迹检测。孵育过一抗(Rabbit anti-HA(CST),Mouse anti-Flag(Sigma),anti-pan-ADP-ribose binding reagent(Millipore))的膜再用偶联HRP的相应二抗(goat anti-mouse(Sigma),goat anti-rabbit(sigma))孵育后，在Amersham Imager 600(GE Healthcare)机器中用ECL发光液(Tanon)检测相应蛋白信号。
3.2.实验结果(如图3所示)
(A)图为WB方式检测HeLa细胞内TDP-43发生PARylation的能力。免疫沉淀法富集外源转染的TDP-43-HA，用PAR的抗体并未检测到发生修饰的TDP-43(箭头指示)。加入双氧水激活细胞内PARP酶活性，提高细胞内PARylation水平的条件下，依然未检测到发生PARylation的TDP-43或者能与TDP-43相互作用的发生PARylation的其它蛋白。以上结果提示TDP-43在体内难以发生PARylation。*代表抗体重链。
(B)图为WB方式检测HeLa细胞内hnRNP A1发生PARylation的能力。通过IP法富集外源转染的hnRNP A1-Flag，能够检测到hnRNP A1在生理情况下发生微弱的PARylation修饰(箭头指示)，进一地提高细胞内PARylation水平不仅可以显著提高hnRNP A1本身PARylation水平，还可以检测到更多与hnRNP A1相互作用发生PARylation修饰的蛋白(#表示)。
(C)在双氧水刺激下，细胞中hnRNP A1自身具有较高水平的PARylation修饰，而且有较多的发生PARylation修饰的蛋白与之相互作用。但是，如果在IP体系中加入PARG蛋白处理1h后，无论是hnRNP A1自身PARylation水平还是与它相互作用的发生了PARylation修饰的蛋白都明显降低，说明在WB中检测到的信号的确是由于PARylation引起的。
(D)hnRNP A1蛋白的功能域示意图。人源hnRNP A1包含两个RNA识别序列(RNA recognition motif,RRM)和位于C端的甘氨酸富集区(Glycin-rich domain,GRD)。示意图上方具体的序列为hnRNP A1潜在的PARylation位点Lys298(加粗标记)，本实施例中将该位点突变为Ala(下划线标记)，后续描述简称为K298A；示意图下方具体序列为潜在的位于两个RRM之间的PAR结合位点(PAR-binding motif,PBM)，该结构域氨基酸序列具有保守性(PBM consensus)，“h”代表疏水氨基酸，“b”代表碱性氨基酸，在本实施例中对该结构域的三个关键氨基酸(加粗标记)进行突变(下划线标记)，在后续实验中记为PBM mut。
(E)图为WB检测细胞内WT,K298A,PBM mut三种hnRNP A1发生PARylation以 及PAR结合的能力差异。与WT相比，K298A突变显著降低hnRNP A1自身PARylation的水平(箭头指示)，不过与PAR结合的能力没有明显变化(#指示)；而PBM mut使得hnRNP A1与PAR结合的能力被破坏，但是自身PARylation的水平却有一定程度的升高。Tubulin作为内参证明三组样品的蛋白上样量一致。以上结果说明hnRNP A1通过PBM与PAR的结合可能会抑制K298位发生过量PARylation修饰。
由本实施例可见，hnRNP A1不仅自身可以发生多聚ADP核糖基化修饰，而且可以通过结合PAR与其它发生此类修饰的蛋白进行相互作用。
实施例4.PARylation或者PAR结合影响hnRNP A1的亚细胞定位和应激响应
4.1.实验步骤
同实施例1-3。
4.2.实验结果(如图4所示)
(A)免疫染色检测WT,K298A,PBM mut三种hnRNP A1蛋白在HeLa细胞中的亚细胞定位及响应细胞压力的情况。图中第一个通道(红色)信号代表TIAR蛋白，作为应激颗粒的标志物；第二个通道(绿色)信号代表外源表达的hnRNP A1蛋白。在无外界刺激条件下(PBS)，WT和K298A几乎全部定位在细胞核内。PBM mut虽然主要定位在细胞核内，但有一定量的蛋白形成异常胞浆点状物(带尾箭头)。在亚砷酸钠刺激下，WT会从核内转移到胞浆中，并与应激颗粒共定位(箭头)。转染K298A的细胞虽然能正常形成应激颗粒，但是突变后的hnRNP A1几乎难以出核(*)。而PBM mut在刺激情况下，虽然也有少量蛋白能够正常进入应激颗粒(箭头)，但仍有大量的异常胞浆点状物形成，该点状物不能与应激颗粒共定位(带尾箭头)。
(B-E)图为对(A)图中能正常形成应激颗粒的细胞的百分比(B)、形成含有hnRNP A1的应激颗粒的细胞百分比(C)、形成胞浆内异常点状物的细胞百分比(D)、以及各个细胞内形成的异常胞浆点状物的数目(E)的统计。以上结果说明，K298位发生PARylation可能是hnRNP A1的出核信号，而通过PBM与PAR的结合能帮助hnRNP A1在亚砷酸钠刺激下向应激颗粒转运。
图4中所有实验均重复三次，每组统计大约300个细胞。非配对、双侧Student’s t-test方法用于相同基因型的亚砷酸钠刺激前后的p value测定；双因素方差分析用于不同基因型之间刺激响应差异的比较。其中，ns代表两组间无显著差异；*为p<0.05,**为p<0.01，***为p<0.001。
实施例3和4的结果说明hnRNP A1致病蛋白发生共价PARylation修饰会影响其亚 细胞定位，而通过非共价结合PAR会影响hnRNP A1蛋白在细胞应激条件下向应激颗粒的转运。hnRNP A1引发的ALS疾病与其异常亚细胞定位和应激响应密切相关(参见“背景技术”)。因此，这部分数据提供了改善PARylation水平以及发展PARP抑制剂用于治疗ALS疾病的细胞调控机理和实验基础。
实施例5.PARylation调节hnRNP A1与TDP-43的相互作用
5.1.实验步骤
同实施例3。
5.2.实验结果(如图5所示)
(A)水解PAR可以减少hnRNP A1与TDP-43的相互作用：免疫共沉淀法检测hnRNP A1能够和内源的TDP-43相互作用；进一步在IP体系中加入PARG水解多聚ADP核糖基团，能够共沉淀得到的内源性的TDP-43蛋白量显著降低。
(B)对(A)图中免疫共沉淀得到的内源TDP-43蛋白相对表达量的统计。
(C)提高PARylation水平可以增强hnRNP A1与TDP-43的相互作用：利用双氧水刺激加si-PARG的双重处理提高细胞内PARylation水平，可以提高与hnRNP A1免疫共沉淀得到的TDP-43的蛋白。
(D)对(C)图中免疫共沉淀得到的内源TDP-43蛋白相对表达量的统计；
图5中所有实验均重复3-5次。统计学差异的显著性通过非配对、双侧Student’s t-test方法检验p value测定，***为p<0.001。
实施例6.PAR促进hnRNP A1在体外发生液-液相分离(LLPS)
6.1.实验步骤
6.1.1蛋白表达和纯化
hnRNP A1蛋白表达所用质粒为pET9d-hnRNP A1(Addgene)，该质粒被表达在E.coli BL21(DE3)pLysS(TranGeneBiotech)中。经过0.4mM IPTG(SangonBiotech)在25℃条件下诱导表达15h后。所得菌通过离心方式收取，加入裂解液(50mM Tris-HCl at pH 7.5,2mM DTT,1mM PMSF,5％glycerin,and 0.1mg/mL RNase A)裂解。裂解产物4℃高速离心取上清，在AKTA(GE Healthcare)机器中上到5ml SP柱中。挂柱的蛋白用buffer A(50mM Tris-HCl pH 7.5,2mM DTT and 5％glycerin)和buffer B(buffer A with 1M NaCl)的混合物(9:1)洗脱。洗脱得到的蛋白用Superdex 7516/600column(GE Healthcare)继续纯化。纯化所得的蛋白溶解于储液buffer(50mM Tris-HCl pH 7.5,500mM NaCl and 2mM DTT)中以用于后续实验。
6.1.2体外LLPS实验
纯化所得的hnRNP A1蛋白与一定浓度的PAR(Trevigen)在一定盐浓度下混合，反应体系为50mM Tris-HCl,pH 7.5,10％(w/v)PEG 3550(Sigma)and 2mM DTT。混合物室温孵育3min后，取5ul滴在载玻片上借助SP8共聚焦显微镜(Leica)进行观察。
6.2.实验结果(如图6所示)
(A)hnRNP A1能自发通过LLPS形成液滴：图为在特定蛋白浓度和盐离子浓度下hnRNP A1通过相变形成液滴的情况，随蛋白浓度的增加和盐离子浓度的降低hnRNP A1液滴的直径和数目逐渐增大。
(B)在临界条件下加入不同浓度的PAR可以促进hnRNP A1发生LLPS，并且随PAR浓度的增加促进能力逐步提高。
(C)高浓度的肝素(Heparin)不能促进hnRNP A1发生LLPS。
(D)高浓度的PAR自身不能发生LLPS。以上结果说明PAR对于hnRNP A1的LLPS促进作用是特异性的。
实施例7.hnRNP A1能够和TDP-43发生共相分离，并且PAR能够促进这一过程
7.1.实验步骤
7.1.1质粒获得
用于体外表达纯化所用的质粒pET9d-hnRNP A1从Addgene购买。另外两个质粒pET-28a-TDP-43 1-274-6×His和pET-28a-TDP-43 274-414-6×His(pET28a质粒来源：Addgene)构建步骤基本同实施例1，插入位点为BamHI和XhoI。所用引物如下：
pET-28a-TDP-43 1-274-6×His对应的引物：
TDP-43 1-274的上游引物:
Figure PCTCN2019074579-appb-000015
TDP-43 1-274的下游引物:
Figure PCTCN2019074579-appb-000016
pET-28a-TDP-43 274-414-6×His对应的引物:
TDP-43 274-414的上游引物:
Figure PCTCN2019074579-appb-000017
TDP-43 274-414的下游引物:
Figure PCTCN2019074579-appb-000018
7.1.2蛋白表达纯化
TDP-43 1-274：表达质粒为pET-28a-TDP-43 1-274-6×His，该质粒被表达在BL21E.coli(DE3)(TranGeneBiotech)中，经过50uM IPTG(SangonBiotech)在19℃条件下诱导表达16h后。所得菌通过离心方式收取，加入裂解液(50mM Tris-HCl,500mM NaCl,pH 8.0,10mM imidazole,4mMβ-mercaptoethanol,1mM PMSF,and 0.1mg/mL RNase A)裂解。裂解产物低温离心后取上清，用Ni柱富集纯化(GE Healthcare)。Ni柱富集蛋白用洗脱液(50mM Tris-HCl,500mM NaCl,pH 8.0,250mM imidazole and 4mMβ-mercaptoethanol)洗脱，再用Superdex 200 1616/600column(GE Healthcare)继续纯化。纯化所得的蛋白最终溶解于储存buffer(50mM Tris-HCl pH 7.5,300mM NaCl and 2mM DTT)以用于后续实验。
TDP-43 274-414：该蛋白被表达在BL21 E.coli(DE3)(TranGeneBiotech)中，加入50uM IPTG(SangonBiotech)在37℃条件诱导表达于包涵体中。收集到的菌用变性裂解液裂解(50mM Tris-HCl,pH 8.0and 6M guanidine hydrochloride)。裂解产物于4℃高速离心1h后取上清，再用Ni柱纯化。蛋白用变性洗脱溶液(50mM Tris-HCl at pH 8.0,6M guanidine hydrochloride and 50mM imidazole)洗脱后，再用HPLC(Agilent)继续纯化，得到的产物用冷冻干燥机(Thermo Fisher)冻干后备用。
7.1.3荧光标记蛋白
纯化所得的TDP-43 1-274和hnRNP A1蛋白(实施例6中所得)分别稀释到脱盐反应液中(50mM Tris-HCl,pH 7.5,500mM NaCl and 4mM Tris(2-Carboxyethyl)Phosphine(TCEP)(Invitrogen))，用脱盐柱除去DTT(GE Healthcare)。除盐后TDP-43 1-274和hnRNP A1蛋白分别加入5倍体积AlexaFluor-555 C2-malemide(Invitrogen)或者AlexaFluor-647 C2-malemide(Invitrogen)室温共孵育2h。标记好的蛋白用Superdex 75 16/600 column(GE Healthcare)继续纯化后备用。
7.1.4体外LLPS实验
纯化所得的hnRNP A1与TDP-43以相应的蛋白浓度在一定盐浓度下混合，反应体系为50mM Tris-HCl pH 7.5,和2mM DTT。混合物室温孵育3min后，取5μl滴在载玻片上借助SP8共聚焦显微镜(Leica)进行观察。
7.2.实验结果(如图7所示)
(A)在图示的蛋白和盐离子浓度下，hnRNP A1并不能自发形成液滴。
(B-E)图为在指定条件下，hnRNP A1分别和BSA(B)，TDP-431-274(C)，或者TDP-43274-414(D-E)发生共相变的情况。(B’-E’)图为在与(B-E)图相同条件下BSA(B’)，TDP-431-274(C’)，或者TDP-43274-414(E’)发生相变能力的检测。以上结果说明，在本实施例的条件下，hnRNP A1仅能与TDP-43 1-274发生共相变。
(F)荧光标记的蛋白验证(A,C,C’)图(黑色框)反应条件下hnRNP A1与TDP-43 1-274发生共相变。第一个通道(红色)代表hnRNP A1，第二个通道(绿色)代表TDP-43 1-274，仅有在二者混合后才能形成液滴，并且两种液滴能够很好的共定位。以上结果说明，hnRNP A1和TDP-43 1-274混合后形成的液滴是共相变的结果，而非一方对另一方的单独促进。
(G)PAR可以促进hnRNP A1和TDP-43 1-274发生共相分离，并且促进程度与PAR浓度呈正相关。
(H)高浓度的PAR不能促进hnRNP A1与TDP-43 274-414发生共相变。
(I)向hnRNP A1或TDP-43 1-274中加入高浓度的PAR不能促使它们发生相变。
(J)荧光标记的蛋白验证(G)图指定条件下(黑色框)PAR对于hnRNP A1和TDP-431-274共相变的促进作用。
实施例6和7说明PAR的加入能够促进hnRNP A1与TDP-43共相分离，而蛋白体外发生共相分离是介导细胞内应激颗粒形成的分子机制(详见“背景技术”)。该体外实验结果与实施例5的细胞实验结果互相印证，说明了提高PARylation水平会促进hnRNP A1与TDP-43相互作用、加强应激颗粒的组装，是阐明PARP抑制剂通过减轻蛋白间相互作用维持应激颗粒动态性、防止其向ALS疾病中出现的不可溶蛋白聚积体发展的分子机制的基础。
实施例8.在类运动神经元NSC-34细胞中过表达hnRNP A1或者TDP-43会引起细胞 毒性
8.1.实验步骤
8.1.1细胞培养
293T细胞在含有10％FBS(Biowest)的DMEM(sigma)中培养。NSC-34细胞用含有10％FBS的RPMI 1640medium(Gibco，11875-093)培养。细胞生长条件为37℃，5％二氧化碳。所有细胞均由ATCC购买。
8.1.2 TUNEL染色
TUNEL染色是借助TMR red in situ Cell Death Detection Kit(Sigma-Aldrich)完成的， 具体步骤按试剂盒中的说明书进行。
8.1.3病毒生产和侵染
借助PolyJet TM转染试剂同时在293T细胞中转染慢病毒的包装质粒和对应的外源hnRNP A1或TDP-43表达质粒。转染48小时后，细胞培液用0.45μm syringe filter(Millipore)过滤收集并用Lenti-X TMConcentrator(Clontech)浓缩，浓缩产物用于后续NSC-34细胞的侵染。
8.1.4细胞活性测定
NSC-34细胞提前一天种在96孔板中(Corning)，并加入相应的慢病毒侵染。侵染后48-72h，用Cell Counting Kit-8(CCK-8)(Dojindo)试剂盒测定细胞活力。简单来说就是将10μL CCK-8溶液加入每个孔中。37℃孵育2.5h后用Synergy2 microplate reader(BioTek Instruments)检测450nm处的吸收光，并按照说明书计算细胞活力值。
8.2.实验结果(如图8所示)
(A-B)过表达hnRNP A1引起NSC-34细胞毒性：明场显微镜观察过表达hnRNP A1的NSC-34细胞形态发生异常，并有明显的细胞死亡发生(A).CCK-8检测hnRNP A1过表达引起细胞活力的下降，并且随着用于过表达hnRNP A1的lenti病毒量的增加，细胞活力进一步下降(B)。
(C-F)过表达TDP-43引起NSC-34细胞毒性：过表达TDP-43引起NSC-34细胞形态的异常(C)及剂量依赖的细胞活力下降(D)。TUNEL染色检测显示过表达TDP-43可以引起细胞死亡(F)。图中第一个通道代表(绿色)TDP-43；第二个通道(红色)为TUNEL信号，说明该细胞已经死亡；第三个通道(蓝色)为DAPI，指示细胞核。
图8中所有实验均重复3次。统计学差异的显著性通过非配对、双侧Student’s t-test方法检验p value测定，ns代表两组间无显著差异；*为p<0.05，**为p<0.01。
实施例9.PARylation水平能够调节由于hnRNP A1和TDP-43引起的细胞毒性
9.1.实验步骤
9.1.1 siRNA转染
siRNA转染步骤同实施例1，转染时间为48-60h。相应的siRNA序列如下：
si-mPARG:5’-GCAGUUUCUUACACCUAUATT-3’(SEQ ID NO.17)
si-mPARP1:5’-CGACGCUUAUUACUGUACUTT-3’(SEQ ID NO.18)
9.1.2 qPCR检测
对于NSC-34细胞总mRNA的提取，相应处理后的细胞被收集起来，加入Trizol(Invitrogen)充分裂解后，依次加入氯仿抽提、异丙醇沉淀得到mRNA后,加入DNase(Promega)消除基因组DNA的污染。取1μg mRNA用High-Capacity cDNA Reverse Transcription Kit(Applied biosystems)进行逆转录。得到的cDNA按照一定比例与SYBR Green qPCR Master Mix(Bimake)、目的基因引物混合后，进行qPCR检测(QuantStudio TM6 Flex Real-Time PCR system(Life Technologies))。所用引物如下：
mParg:
上游引物：5’-AGCCTCTGACACGCTTACAC-3’；(SEQ ID NO.19)
下游引物：5’-CAGTCACACCACCTCCAACA-3’(SEQ ID NO.20)
mGAPDH:
上游引物：5’-CACCATCTTCCAGGAGCGAG-3’；(SEQ ID NO.21)
下游引物：5’-CCTTCTCCATGGTGGTGAAGAC-3’(SEQ ID NO.22)
9.2.实验结果(如图9所示)
(A-C)敲低PARP1能缓解hnRNP A1或TDP-43引起的细胞毒性。NSC-34细胞中侵染hnRNP A1(A)或者TDP-43(B)会导致细胞活力显著降低，而这种降低会在敲低PARP1(si-PARP1)后得以缓解。(C)图为使用WB实验检测使用siRNA敲低PARP1蛋白表达水平的效果以及降低细胞中PARylaiton水平的情况。GAPDH作为内参证明两组样品的蛋白上样量一致。以上结果说明，通过遗传学手段降低细胞内PARylation水平能够缓解hnRNP A1和TDP-43引起的细胞毒性。同时该结果提示，PARP1在PARylation修饰对hnRNP A1和TDP-43的调控中起关键作用。
(D-F)敲低PARG会加重hnRNP A1或TDP-43引起的细胞毒性。分别用WB的方式检测使用si-PARG提高细胞整体PARylation的水平及qPCR的方式检测使用si-PARG敲低PARG的mRNA水平的情况(D)。在NSC-34细胞中敲低PARG会进一步加重hnRNP A1(E)或者TDP-43(F)引发的细胞毒性。以上结果说明，通过基因学手段提高细胞内PARylation水平会加重hnRNP A1和TDP-43引起的的细胞毒性。
(G)PARP抑制剂Olaparib能够缓解TDP-43引起的细胞毒性。5μM Olaparib可以降低TDP-43过表达引起的细胞毒性。该结果说明，在药理学水平上降低细胞内PARylation水平能够缓解TDP-43引起的细胞毒性。
图9中所有实验均重复3次。统计学差异的显著性通过非配对、双侧Student’s t-test方法检验p value测定，ns代表两组间无显著差异；*为p<0.05，**为p<0.01，***为p<0.001。
正如背景介绍部分所述，ALS疾病的一个主要特征是运动神经元的死亡和丢失。本发明利用一种类运动神经元细胞NSC-34作为模型来研究hnRNP A1和TDP-43所导致的细胞毒性对运动神经元的影响。在NSC-34细胞中过表达hnRNP A1或TDP-43可以引发类似ALS疾病中这两个致病蛋白对细胞产生的异常影响，如细胞形态的变化、细胞凋亡过程中DNA的断裂(通过TdT-mediated dUTP Nick-End Labeling(TUNEL)染色法检测)和细胞数量和活力的改变(通过Cell Counting Kit-8(CCK-8)法检测)等。细胞出现死亡或者活力降低，说明hnRNP A1或TDP-43蛋白的异常会产生细胞毒性，对应到ALS疾病中意味着这两种蛋白的异常可能会使得运动神经元死亡，最终导致ALS疾病的发生。而实施例8和9的结果表明，无论通过遗传性方法还是药理学方法减少PARP的水平或活性，均可以有效地抑制hnRNP A1和TDP-43过表达导致的对类运动神经元NSC-34细胞的毒性。这一结果能够最直接、最有力的提示PARP抑制剂有望延缓ALS病人的运动神经元死亡。
实施例10.在ALS果蝇模型中下调PARP可以减弱由于TDP-43引起神经退行性病变
10.1.实验步骤
10.1.1果蝇获得
本发明所用果蝇来自Bloomington Drosophila Stock Center(BDSC)：RNAi-Parp(#57265),elavGS(#43642),RNAi-mCherry(#35785,实验中所用的RNAi-ctrl果蝇)，GMR-GAL4(#79573)。UAS-TDP-43果蝇为本实验室借助ΦC31转座酶在果蝇基因组中定点插入所得。相应的驱动子果蝇与特定的RNAi或UAS果蝇交配即可产生符合要求的子代。所有实验用果蝇都饲养于标准玉米粉培养基中，饲养温度为25℃，湿度为60％。实验所用的果蝇均为雄性果蝇，完整基因型如下：
用于果蝇mRNA水平检测时的完整基因型：
w；UAS-RNAi-mCherry/GMR-GAL4
w；UAS-RNAi-Parp/GMR-GAL4
用于果蝇视神经退化及外源表达蛋白检测时的完整基因型：
w；UAS-hTDP-43/+；UAS-RNAi-mCherry/GMR-GAL4
w；UAS-hTDP-43/+；UAS-RNAi-Parp/GMR-GAL4
用于果蝇爬行能力和生存能力检测时的完整基因型：
w；UAS-hTDP-43/+；UAS-RNAi-mCherry/elavGS
w；UAS-hTDP-43/+；UAS-RNAi-Parp/elavGS
10.1.2蛋白提取
对于果蝇蛋白的提取，相应基因型的果蝇的头部被分离下来，加入RIPA裂解液(50mM Tris pH 8.0,150mM NaCl,1％NP-40,5mM EDTA,0.5％sodium deoxycholate,0.1％SDS,蛋白酶抑制剂)后在冰上充分研磨，裂解后的产物经过四度高速离心后，取上清加入4x LDS sample buffer(Invitrogen)，95℃煮沸。对于不可溶相的获得，RIPA裂解后离心得到的沉淀用与可溶相等体积的9M urea buffer(9M urea,50mM Tris buffer,pH 8.0)裂解后，也加入4x LDS sample buffer(Invitrogen)，95℃煮沸以便后续研究。
10.1.3 qPCR检测
对于果蝇mRNA的提取，相应基因型的果蝇头部被分离下来，加入Trizol(Invitrogen)后，用研磨杵充分研磨后，加入氯仿抽提、异丙醇沉淀得到mRNA后,加入DNase(Promega)消除基因组DNA的污染。取1μg mRNA用High-Capacity cDNA Reverse Transcription Kit(Applied biosystems)进行逆转录。得到的cDNA按照一定比例与SYBR Green qPCR Master Mix(Bimake)、目的基因引物混合后，进行qPCR检测(QuantStudio TM6 Flex Real-Time PCR system(Life Technologies))。所有引物如下：
dParp:
上游引物：5’-ATGAAGTACGGAGGCCAACC-3’(SEQ ID NO.23)；
下游引物：5’-TCTTCACCTGACGCAAACCA-3’(SEQ ID NO.24)
dActin:
上游引物：5’-GAGCGCGGTTACTCTTTCAC-3’(SEQ ID NO.25)；
下游引物：5’-GCCATCTCCTGCTCAAAGTC-3’(SEQ ID NO.26)
10.1.4果蝇外眼损伤、爬行和生存能力检测
果蝇外眼的观察：相应基因型的果蝇在羽化5天和20天后在SZX16(Olympus)显微镜下观察并拍摄其外眼图片。每只果蝇根据其外眼损伤程度依次记作“0，1，2，3，4”5个不同等级，其中“0”代表完全无损伤，“4”代表完全损伤。每只果蝇作为一个独立样本参与后续统计。
果蝇爬行能力检测：在果蝇羽化的第一天，挑出相应基因型的果蝇，每20只一组饲养于含有80μg/ml RU486(TCI)的食物中以诱导转基因表达。在指定日期，将果蝇按20只一组分至5-8个透明塑料管中，轻轻振动塑料管，将果蝇震到管底，开始计时，并记录在10s内能爬过3cm高度的果蝇的数目，计算其百分比(每管中果蝇整体作为一个独立样本参与后续统计，下同)。所有实验重复三次。
果蝇生存能力检测：在果蝇羽化的第一天，挑出相应基因型的果蝇按20只一组分为 7-9管，饲养于含有80μg/ml RU486(TCI)的食物中。所有用于生存能力检测的果蝇每三天换一次加药的新鲜食物，记录每次各管中死亡的果蝇数目，计算果蝇的存活率(意外飞走的果蝇不计入总数)。
10.2.实验结果(如图10所示)
(A-E)在果蝇体内敲低PARP能缓解TDP-43转基因引起的视神经退化。(A)图为实验中单只代表性果蝇的外眼图片。用GMR驱动子在果蝇的光感受器神经中表达(按所列出的基因型交配果蝇品系，转基因序列即可在子代果蝇的特定细胞中自动表达。此步骤按照本领域常规的分子遗传学基本操作即可)人源TDP-43蛋白(按所列出的基因型交配果蝇品系，其中编码人源TDP-43蛋白的转基因即可在果蝇体内表达；TDP-43蛋白序列如SEQ ID NO.2所示)会造成明显的视神经退化，主要表现为色素的丢失和外眼表面的不光滑，并且这种表型会随果蝇年龄的增加而加重(Day5vs Day20)。而在该转基因果蝇中同时表达RNAi-Parp(同上，按所列出的基因型交配果蝇品系，转基因序列即可在子代果蝇的特定细胞中自动表达)可以很好地缓解由于TDP-43造成的神经退行性病变。左上角图片为白色框中区域的放大。(B)对(A)图中外眼损伤程度的统计。括号中的数字代表参与统计的果蝇数目。(C)qPCR检测RNAi-Parp转基因果蝇头部Parp的mRNA水平。(D)图为使用WB验证敲低Parp对TDP-43转基因果蝇的外源蛋白表达量及可溶性均无明显的影响。其中GAPDH作为内参证明两组样品的蛋白上样量一致以及不可溶相中无可溶相蛋白的污染。(E)对(D)图中TDP-43蛋白相对表达量的统计。“ud”表示未能检测到蛋白。
(F-G)在成年果蝇神经元中敲低PARP能够改善人源TDP-43表达引起的爬行能力缺陷和寿命缩短：(G)在elavGS驱动下，在成年的果蝇的神经元中诱导人源TDP-43转基因的表达会引起年龄依赖的爬行能力的丧失,而同时敲低PARP能够缓解这一表型。(F)相同条件下的果蝇寿命与对照组相比有明显缩短，敲低PARP能够一定程度上延长转基因果蝇的寿命。括号中的数字分别代表最终统计的果蝇数目和半数存活天数。
图10中所有实验均重复3次。统计学差异的显著性通过非配对、双侧Student’s t-test方法检验p value测定，ns代表两组间无显著差异；**为p<0.01，***为p<0.001。
实施例10的结果表明，不仅在体外和细胞实验中，而且在ALS体内动物模型和生命体整体水平上，减少PARP酶的水平对于抑制TDP-43过量导致的神经元形态、功能的退行性病变乃至寿命的急剧缩短都有明显的改善效果，提示着发展PARP抑制剂够治疗ALS疾病具有良好的前景。
本发明中的PARP抑制剂Olaparib是FDA批准的用于治疗乳腺癌、卵巢癌和前列腺癌等肿瘤的临床用药(Fong et al.,2009；Tutt et al.,2010)，其毒副作用已经经过临床论证。同时，本发明显示该抑制剂对于hnRNP A1和TDP-43引发的神经细胞毒性均有明显抑制作用，故该抑制剂用于开发ALS临床用药的可能性更大、风险更小。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (12)

  1. 一种治疗神经退行性疾病的药物组合物，其特征在于，所述药物组合物包括降低细胞内多聚ADP核糖基化修饰水平的药物，优选降低细胞内RNA结合蛋白的多聚ADP核糖基化修饰水平的药物；所述RNA结合蛋白优选为hnRNP A1和/或TDP-43。
  2. 如权利要求1所述的治疗神经退行性疾病的药物组合物，其特征在于，所述神经退行性疾病为肌萎缩性脊髓侧索硬化症。
  3. 一种治疗RNA结合蛋白发生异常导致的疾病的药物组合物，其特征在于，所述药物组合物包括降低细胞内多聚ADP核糖基化修饰水平的药物，优选降低细胞内所述RNA结合蛋白的多聚ADP核糖基化修饰水平的药物；所述RNA结合蛋白优选为hnRNP A1和/或TDP-43。
  4. 如权利要求3所述的治疗RNA结合蛋白发生异常导致的疾病的药物组合物，其特征在于，所述治疗RNA结合蛋白发生异常导致的疾病包括以下一种或多种：
    (a)RNA结合蛋白的异常翻译后修饰引起的细胞内异常聚集导致的疾病；
    (b)RNA结合蛋白的异常亚细胞定位导致的疾病；
    (c)RNA结合蛋白所参与的应激颗粒的形成和/或调控发生异常导致的疾病。
  5. 如权利要求3所述的治疗RNA结合蛋白发生异常导致的疾病的药物组合物，其特征在于，所述治疗RNA结合蛋白发生异常导致的疾病包括以下一种或多种：
    (a)RNA结合蛋白结合和/或调节RNA的加工、剪切、转运和/或翻译发生异常导致的疾病；
    (b)RNA结合蛋白如hnRNP A1发生共价的多聚ADP核糖基化修饰影响所述RNA结合蛋白如hnRNP A1与其它蛋白如TDP-43蛋白的相互作用和/或亚细胞定位导致的疾病；
    (c)RNA结合蛋白如hnRNP A1通过非共价结合PAR影响所述RNA结合蛋白如hnRNP A1与其它蛋白如TDP-43蛋白的相互作用导致的疾病；
    (d)RNA结合蛋白通过非共价结合PAR影响其在细胞应激条件下向应激颗粒的转运导致的疾病；
    (e)RNA结合蛋白通过非共价结合PAR影响其在细胞中的可溶性改变从而引起细胞稳态破坏导致的疾病。
  6. 如前述任一项权利要求所述的药物组合物，其特征在于，所述降低细胞内多聚ADP核糖基化修饰水平的药物为能够提高PARG水解酶表达水平和/或降低PARP聚合酶 表达水平的药物。
  7. 如权利要求6所述的药物组合物，其特征在于，所述能够提高PARG水解酶表达水平的药物为PARG水解酶激动剂；所述能够降低PARP聚合酶表达水平的药物为PARP聚合酶抑制剂；所述PARP聚合酶抑制剂优选Olaparib。
  8. 一种治疗如权利要求1或2中所述的神经退行性疾病、或如权利要求3～7任一项中所述RNA结合蛋白发生异常导致的疾病的方法，其特征在于，所述方法包括利用如权利要求1～7任一项所述的药物组合物进行治疗。
  9. 如权利要求8所述的方法，其特征在于，所述药物组合物通过以下的一种或多种机制进行治疗：
    (a)影响hnRNP A1和/或TDP-43形成应激颗粒，优选抑制应激颗粒的组装和/或促进应激颗粒的去组装；
    (b)影响hnRNP A1和其它多聚ADP核糖基化修饰的蛋白之间的相互作用；
    (c)影响hnRNP A1和其它PAR结合蛋白之间的相互作用；
    (d)影响hnRNP A1的亚细胞定位和/或应激响应；
    (e)影响hnRNP A1的液-液相分离和/或聚集倾向；
    (f)影响hnRNP A1和TDP-43蛋白的共相分离；
    (g)影响hnRNP A1和TDP-43蛋白在细胞中的相互作用；
    (h)抑制hnRNP A1和/或TDP-43引起的神经细胞毒性。
  10. 如权利要求9所述的方法，其特征在于，所述药物组合物最终可以抑制hnRNP A1和/或TDP-43所引起的神经变性；
    较佳地，所述神经变性包括：1)在类运动神经元NSC-34细胞中的毒性抑制作用，和/或，2)对ALS疾病果蝇模型中神经元退化、运动能力降低和/或寿命缩短的缓解作用。
  11. 如权利要求1～7任一项所述的药物组合物在治疗如权利要求1或2中所述的神经退行性疾病、或如权利要求3～7任一项中所述RNA结合蛋白发生异常导致的疾病中的应用。
  12. 如权利要求1～7任一项所述的药物组合物在制备治疗如权利要求1或2中所述的神经退行性疾病、或如权利要求3～7任一项中所述RNA结合蛋白发生异常导致的疾病的药物中的应用。
PCT/CN2019/074579 2019-02-02 2019-02-02 治疗神经退行性疾病或rna结合蛋白发生异常导致的疾病的药物组合物及其应用 WO2020155141A1 (zh)

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